Difference between revisions of "Team:USTC/Results"

Latest revision as of 16:20, 14 October 2015

Overview
Permeability Improvement
Chemotaxis & ROSE
Adhesion Assay
Film Determination
Calibration

That is so amazing! We finally made NDM this year! The results page will deliver the fresh results from us. Our results include these sections:

Permeability Improvement: In this section, we will demonstrate our synthetic bioloical base constrcuction, characterization of permeability improvement. This is the fundation of CACCI construction, as well as NDM.
Chemotaxis Modification: This section introduces results of chemotaxis engineering.
Adhesion Assay: This section explicitly explain adhesion methods, adhesion dynamics and adhesion protocols recommended for all users, which is an important part of our results.
Film Determination: This section introduced how we finally determinated our film, from plastic film to our final candidate. See how difficult and interesting it is!
Calibration: This is our final step demonstrating the feasibility of NDM. See how perfect our project is!

Extraction of Permeability Improvement Fragements

We successfully get gene fragements that are used for improving bacterial permeability, which including,

SCVE, a viroporin originally from SARS virus, is synthesized by Sangon Co. ctd.

OprF, a bigger porin compared with OmpF in E. coli, is extracted from P.aeruginosa， PAO1 genome by PCR.

T7 is a strong promoter which we got form Parts Registry. T7 will be used to trigger the expression of both OprF and SCVE.

Figure 3: T7 extraction from parts
Cas9 is got from 2015 Part Distribution.

gRNA-AcrB and gRNA-EmrE, these two fragments are for silencing bacterial transmembrane protein AcrB and EmrE to strongly block drug efflux system. These two parts are synthesized by Sangon Biotech Company.

Figure 5: Extraction of gRNA-AceB and gRNA-EmrE, two fragments are synthesized by Sangon Biotech Company

Construction of Permeability Improving Plasmid

Construction of plasimid with T7-SCVE (BBa_K1593667) . We finally ligated the T7 with SCVE and got T7-SCVE (BBa_K1593667).

Figure 6: Construction of T7-SCVE ([BBa_K1593667](http://parts.igem.org/Part:BBa_K1593667))

Construction of plasimid containing T7-OprF (BBa_K1593210). This is the result of T7 with OprF.

Figure 7: Construction of T7-OprF ([BBa_K1593210](http://parts.igem.org/Part:BBa_K1593210))

Growth Characterization

We firstly characterized the growth rate of genetically modified CACCI along with wild type BL21 using optical density at 600 nm, which is called OD 600, in order to demonstrate that the overexpression of porin(OprF with T7,BBa_K1593209) or viroporin(SCVE with T7, (BBa_K1593667) won't severely affect the growth of bacteria, at least it won't significantly inhibit bacteria growth and become a kill switch.

All bacteria are cultured previously in LB medium about 12 h. And then the measurement of bacteria growth through time began at 8 A.M. the other day.

Figure 8: Bacteria growth characterization

As the images above illustrated, we found E. coli BL21 wild type has the best growth characteristics with a maximal OD600=3.427. Whereas E. coli BL21 with OprF (BBa_K1593210) has a relatively low optical density as its OD600 after 12 h is 2.808 compared to the wildtype. In the same way, characterization of E. coli BL21 with SCVE, http://parts.igem.org/Part:BBa_K1593667 showed a slightly smaller OD600, as its finally turned to 2.835. E. coli BL21 with T7-OprF (BBa_K1593210) and E. coli BL21 with T7-SCVE, (BBa_K1593667) are showing 19% decreased maximal cell density and 18.5% decreased maximal cell density respectively in comparison with wild type.

Consequently, the result implies that the genetical modification on bacterial permeability, at least the overexpression of OprF and SCVE with a strong promoter T7, won't significantly influence the bacterial growth. Thus, genetically modified bacteria can be used in the following experiments.

Later on, we will characterize the basic permeability capability of E. coli BL21 with T7-OprF (BBa_K1593210) and E. coli BL21 with T7-SCVE, (BBa_K1593667).

ONPG Assay

ONPG, that is the abbreviation of ortho-Nitrophenyl-β-galactoside. ONPG is a colorimetric and spectrophotometric substrate, previously used for detecting β-galactosidase activity. ONPG is actually colorless at normal situation, however if it triggers the degradation reaction catalyzed by β-galactosidase, we will get galactose and ortho-nitrophenol. Surprisingly, the compound ortho-nitrophenol has a yellow color, which means we are able to detect the chemical reaction through OD detection. Besides, owing to the existence of β-galactosidase in bacteria, a more intense optical density we detected, that means there are more ONPG uptaked by bacteria. Therefore, using ONPG we are able to distinguish the permeability capability of different bacteria.

Here's our result.

Note: The wavelength for detecting the bacterial concentration and absortion of ONPG are respectively 600nm and 406nm.

First, we detected the bacterial solutions to control the initial concentrations.

The OD values for E.coli BL21, E.coli BL21 with T7-OprF (BBa_K1593210) , E.coli BL21 with T7-SCVE(BBa_K1593667) are respectively 2.856,1.806 and 1.889. Based on these, we know that the concentrations of these three bacteria are close.

Then we add ONPG to the bacterial solutions and detect the OD.

From this figure, we can see a rising trend in all bacterial solutions. The OD values for E.coli BL21 with T7-OprF(BBa_K1593210) and E.coli BL21 with T7-SCVE(BBa_K1593667) are respectively 0.2520 and 0.2588,both are higher than E.coli BL21,the wild type. We can infer that the absorbtion of modified bacteria is higher than the wild type.

There is a more visual figure for comparing the absorbtions. We made a zero calibration using the OD value of wild type as the zero level.

Figure 12: ONPG assay, absorption OD406 in average

In general, the OD values of both two modified bacteria are rising. For E.coli BL21 with T7-OprF(BBa_K1593210), the rising is relatively smooth. For E.coli BL21 with T7-SCVE(BBa_K1593667),the value goes down first, then goes up .And the E.coli BL21 with T7-SCVE(BBa_K1593667) has a higher absorbtion than E.coli BL21 with T7-OprF(BBa_K1593210).

NPN Assay

Figure 13: Schematic Structure of N-Phenyl-1-naphthylamine, NPN

N-Phenyl-1-naphthylamine(NPN),C16H13N, CAS:90-30-2, FW: 219.29, is a nonpolar probe, whose fluorescence signal is relatively strong in a phospholipid environment, but weak in aqueous surroundings. Consequently, using this feature, we are able to characterize bacteria permeability by measuring the fluorescence intensity of the bacteria solution. When NPN molecules are absorbed by bacteria, we will observe a significantly rising fluorescence intensity. The more fluorescence intensity we got, the more permeable the bacteria are. Here are what we got from NPN uptake assay and the conclusion on genetically engineered bacteria permeability improvement.

Here's our results, the first picture illustrate the total fluorescence intensity measurement by BMG Labtech CLARIOstar®.

Note: The fluorescence intensity signal at excitation wavelength range is 305nm to 335nm and emission wavelength range is 370nm to 410nm, which is recommended by previous research.

The total fluorescence intensity of E. coli BL21 wild type is 126451. On the other hand, the fluorescence intensity in E. coli BL21 with T7-OprF (BBa_K1593210) and E. coli BL21 with T7-SCVE, (BBa_K1593667) are respectively 207322 and 151820, which are significantly larger than the wildtype assay.

Though we have already got the exact data on NPN uptake, we still need to revise its effectiveness because of the different bacteria concentration of E. coli BL21 wild type, E. coli BL21 with T7-OprF (BBa_K1593210) and E. coli BL21 with T7-SCVE, (BBa_K1593667). Here is the exact bacteria concentration data, based on OD600nm,

Strain	OD600nm
E. coli BL21 wild type	3.663
E. coli BL21 with T7-OprF (BBa_K1593210)	2.431
E. coli BL21 with T7-SCVE, (BBa_K1593667)	2.441

After correcting these data, we are able to get bacteria absorption uptake capability in average, which is a more accurate value to characterize bacterial permeability property.

The fluorescence intensity in average of E. coli BL21 wild type is 34521.16. On the contrary, the fluorescence intensity revised by bacterial concentration in E. coli BL21 with T7-OprF (BBa_K1593210) and E. coli BL21 with T7-SCVE, (BBa_K1593667) are respectively 85282.60 and 62195.8, which are relatively 2.5 folds and approximately 2 folds to E. coli wild type, that is a significantly improvement compared to wild type.

Consequently, through NPN uptake assay, we are able to conclude that the small molecule uptake capability of bacteria improved after our genetical modification. Thus E. coli BL21 with T7-OprF (BBa_K1593210) and E. coli BL21 with T7-SCVE, (BBa_K1593667) are both able to use as the candidate bacteria strains for NDM measurement.

Bibliography

T. Mattila-Sandholm et al. Fluorometric assessment of Gram-negative bacterial permeabilization. Journal of Applied Microbiology 2000, 88, 213–219
Scott Banta et al. Genetic Manipulation of Outer Membrane Permeability: Generating Porous Heterogeneous Catalyst Analogs in Escherichia coli dx.doi.org/10.1021/sb400202s ACS Synth. Biol. 2014, 3, 848−854

Extraction of Permeability Improvement Fragement

Construction of Permeability Improving Plasmid

Growth Characterization

ONPG Assay
NPN Assay
Bibliography

Genetic Fragments Extraction

micF and SoxS, these two antibiotic substance responding promoters were extracted from E. coli Top 10(K-12 strain) by conducting polymerase chain reaction on its genome.

Figure 1: fragements extraction of micF and SoxS

cheZ, this is the chemotaxis modified fragment.

BBa_R0010, this is promoter of lac, which triggers the expression of cheZ.

[Figure 3: fragment extraction of OprF and Lac](http://parts.igem.org/Part:BBa_K1593997)

Construction of plasimid with lac-cheZ BBa_K1593997

This is our final construction of lac-cheZ BBa_K1593997

Figure 4: plasmid construction of lac-cheZ [BBa_K1593997](http://parts.igem.org/Part:BBa_K1593997)

Growth characteristics

We detected OD600(nm) to identify the effect of cheZ (BBa_K1593997) expressed on E. coli BL21 with time induced by IPTG or not.

As the images above illustrated, we found E. coli BL21 wild type has the best growth characteristics with a maximal OD600=2.6. Whereas E. coli BL21 with cheZ (BBa_K1593997) induced by ITPG has a relatively low optical density as its OD600 after 9 h is 2.3 compared to the wildtype. In the same way, characterization of E. coli BL21 with cheZ (BBa_K1593997) not induced by IPTG showed a slightly smaller OD600, as its finally turned to 2.1. The exogenous protein expression affects the growth of bacteria, which was indicated by slight slower grow-up in E. coli containing cheZ plasmid. In total, cheZ doesn't have obvious negative impact on bacteria grown.

SDS-PAGE

After induced by IPTG in 1mM about 4 h, cheZ was found in SDS-PAGE gel, compared to the wild type (Top10) and bacteria without IPTG induction. Through this result, we concluded that cheZ has been successfully expressed with the IPTG induction.

See protocol in Protocols

Genetic Fragments Extraction

Growth characteristics

SDS-PAGE

Characterization of Optimal Conditions on Polylysine(PLL) Coated Assay

To see the details on the mechanism of polylysine adhesion, please refer to Project-CACCI.

To get the final PLL-coated protocol, check Protocols for further information.

Our original characterization of optimal conditions on polylysine(PLL)-coated assay requires several factors to get, including:

Best bacterial developmental interval along with recommended dilution conditions.
Best PLL-Coated Concentration and Pre-treatment Time.
Best PLL-Coated Time, given full consideration to the completion of bacterial adhesion time.
Best Measurement Interval, which illustrates the possible time for bacterial response on antibiotics pressure.
Possible Determination Interval, which refers to antibiotics response interval.

Here we present all the optimistic conditions for users to get the effective results on antibiotics using our CACCI and SPRING.

Best Bacterial Developmental Interval Along with Recommended Dilution Conditions

According to experience on E. coli development based on OD detector. We concluded that bacteria exploding during Logarithmic Period(OD:535nm about 0.4~0.5) are at the most energetic moments with proper bacterial density. Consequently, we highly recommend user to culture bacteria at Logarithmic Period and then dilute bacterial solution about twenty times to fifty times.

Best PLL-Coated Concentration and Pre-treatment Time.

Best PLL-Coated Conditions, which include PLL-Pretreatment time, PLL-Coated concentration and PLL-Coated time are measured for best adhesive condictions for bacterial treatment.

As for PLL-Pretreatment time, we adopt traditional pretreatment time, incubating about 12~16 h, for recommendation, which is originally used for neurons coated on neural science research. And when it comes to PLL-coated concentration, experience on neurons adhesion is also precious. According to previous research, PLL concentration in the interval of 20 ug/mL to 100 ug/mL is quite effective for bacterial adhesion.

Adhesion Assay with PLL treatment

Here we deliver the PLL treatment results comparing with no PLL treatment assay. During pre-experiment adhesion assay, PAO1, a strain of Pseudomonas aeruginosa, is used for adhesion effects.

The picture below showed the amount of bacteria(PAO1) under microscope without PLL treatment, which is abbreviated as PLL(-). To observe capability of bacterial adhesion, we wash observed place with PBS:

Figure 1: Control Assay-Without PLL treatment

After elution, a significant decline in the number of bacteria is recorded. As a matter of fact, there is no bacteria in observed field after PBS washing.

To get the algorithm of bacterial counting, please refer to Modeling: Adhesion Dynamics.

Figure 2: Control Assay-Without PLL treatment2

As for treatment with PLL in 20 ug/mL, which is abbreviated as PLL(+,20), the impressive adhesion effect is observed. To ensure the exact effect, bacteria observing field is washed twice by PBS.

Figure 3: Adhesion Assay-With PLL treatment in 20 ug/mL

The number of bacteria doesn't decline, and on the contrary, we see slight increase of bacteria number. The possible reason is constant bacterial settlement during adhesion assay. After twice PBS wash, the number of bacteria is relatively stable as well. Consequently, we concluded PLL has significant adhesive ability on bacteria.

Figure 4: Adhesion Assay-With PLL treatment in 20 ug/mL

Best PLL-Coated Time

Then, we tried to figure out the cohesive effect of polylysine through time to get the best PLL-coated time. We gathered data just after PLL treatment(0s), after 1min and after 5min. And we also recorded bacteria number in the following 20 s. Here we provide the analysis of bacterial number variation after PLL treatment.We respectively use PAO1, a kind of Pseudomonas aeruginosa and HCB1, a kind of E. coli to handle the assay. PAO1 contains self-adhesive ability and HCB1 has strong mobile ability. Using these genetically natural bacteria, we would conclude with the effect of polylysine treatment.

As for PAO1, Without polylysine coated, bacteria have strong swimming ability. Because PAO1 is a kind of self-adhesive bacteria for experiment, thus we could see adhesive bacteria increasing during assay.

Figure 5: no antibiotics bacterial number variation through time without PLL

With 20 ug/mL polylysine treatement, bacterial adhesive effect due to strong electrostatic adhesion becomes stronger, and we, as well are able to observe the number of adherent bacteria gradually rising until reaching equilibrium.

Figure 6: no antibiotics bacterial number variation through time with PLL

After mathematical simulation we found that the adhesion rate of PAO1 by polylysine after treatment of PLL is increased, while for E. coli, the adhesion quantity rise, not fall.

图片名称

As for HCB1, a kind of E. coli and the number of bacteria inside observed field is stable and relatively declined because of its lack of capability of adhesion.
图片名称

After treated with 20ug/mL polylysine, adhesive bacteria number obviously increased:
图片名称

Modeling on adhesion process strictly demonstrated the adhesion assay fits Langmuir absorption isoform.

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Simulation result:
\(\sigma =K\sigma _{0}\times (1-e^{-\frac{K_{\alpha}CV_{z}}{K\sigma _{0}}(t-t_{0})})\)
Constants value and details:

To know more about our modeling, please check Modeling: Adhesion Dynamics

This significant reverse of bacterial adhesion tendency stronly proved that polylysine has effective adhesive ability for SPRING.

After we confirmed the feasibility of PLL treatment for bacterial adhesion, then the treating time should be taken into consideration because only in the case of stable adhesion is effective for SPRING to gather effective and stable data.

As the plot illustrates, bacteria number is growing at the beginning of PLL treatment because of opening strong electrostatic adsorption derived from PLL. And after 1min, the number of bacteria become stable, and there is nearly no difference on bacteria number comparing after 1 min treatment to after 5 min treatment.

Consequently, we recommended that PLL treatment after 5 min would be a promising set for SPRING to output stable data.

Best Measurement Interval

According to chemotaxis, bacteria responsing to surrounding pressure or beneficits will be presented as change of bacteria movement. Here we analyzed bacterial movement data treating with different antibiotics concentration, specificly, chloromycetin concentration. And we gathered bacterial movement data just after antibiotics treatment(0s), after 1 min treatment and after 5 min treatment, and we again recorded all pictures in the following 18 s to get the dynamic data. Then, we concluded from the percentage of bacterial movement as an important data to intercept the effect of bacterial response on antibiotics.

The measurement assay is illustrated as following:

PLL(-)

Antibiotics Concentration(ug/mL)	Incubation Time(s)
0	0
0	60
0	120
0	180
0	240
0	300
1	0
1	60
1	300

PLL(+,20)

Antibiotics Concentration(ug/mL)	Incubation Time(s)
0	0
0	60
0	120
0	180
0	240
0	300
0.1	0
0.1	60
0.1	300
0.5	0
0.5	60
0.5	300
1	0
1	60
1	300

When bacteria are treated with PLL, the movement of bacteria is been limited for strong adhesion, after 5 min treatment, the proportion of movement decreased through time.
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However, when bacteria are not treated with PLL, incubated in 1ug/mL chloromycetin solution, we got the movement percentage of bacteria as following:

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The amount of moving bacteria is relatively increasing after 5 min antibiotics treatment. Bacause of no PLL treatment, we could see the relative increase of bacteria movement percentage.

As for bacteria treated with 20 ug/mL PLL, different pattern is observed:

This is the bacterial movement percentage variation with time, treated with 0.1 ug/mL chloromycetin solution. Antibiotics pressure is not that strong, consequently, the movement of bacteria is not ignited significantly.

图片名称

When it comes to 0.5 ug/mL chloromycetin solution, the proportion of the moving bacteria is increased. And as a matter of fact, the increase is quite corresponding through time, reflecting bacteria impressive response on antibiotics substance.
图片名称

What if the concentration of chloromycetin solution up to 1 ug/mL?
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A quite linear increase of the proportion of the moving bacteria with time is observed.

Consequently, according to our experiment, genetically naturally bacteria could respond to chloromycetin solution from 0.1 ug/mL to more than 1ug/mL, which is quite promising after genetic modification.

图片名称

Characterization of Optimal Conditions on Polylysine(PLL) Coated Assay

Best Bacterial Developmental Interval Along with Recommended Dilution Conditions

Best PLL-Coated Concentration and Pre-treatment Time.

Adhesion Assay with PLL treatment

Best PLL-Coated Time

Best Measurement Interval

Let's do some crasy science this time! We'll show you how we test and select the most important part in our project, the special artificial original film!

Modeling guide us choose the candidates

In our modeling, we start with single bacteria force, analyse the interaction between bacteria and film, and propose the request of Young modulus (<1GPa) of film eventually.

And there are various film candidates come into the front.

Processing Film

The film must be kind of soft and one of the surface should have enough reflectance while another surface should have the ability to adhere to bacteria.
In fact, we don't hear any of this type of film. So we need to produce the film by our own.

Film I

Low Pressure Polyethylene

Low pressure polyethylene is soft enough, and is too soft.

We use aerosol paint cover one of the surface. That can make the other surface of the film will become a reflect surface. And we use 400ul 20ug/ml PLL coating the same surface at the temperature of 4℃ to make this surface has the ability to adhere to bacteria.

But we fail with film I in the end that we can't get interference fringes. Because the surface of low pressure polyethylene is not as smooth as we want, that will cause a diffuse reflection. Thus we can not get interference inescapably.

So we need the film becoem more smooth and more elastic, and then come out film II-Rubbers

Film II

Rubbers-Condom

If you want the material smooth enough and thick enough and elastic enough, that is, of cause, condom!

When we plan to use paint cover, the truth give us a hard hit. Because the film shrink too much when we spray paint.

And we try silver mirror reaction as replacement. But still fail Because the ammonia erosion is too severe.

Finally we find the final film candidate that we missed-Glass

Film III

Cover slip

The Young modulus of Glass is about 50GPa. But according to our adhesion assay result and modeling, there will several numbers of fringes changes in the experiment.

So we spraying it, coating it, testing it, and we made it!
The glass have a great potential to reflect, so we can get interference very easy.
With modeling guidance, we can know the relation between fringes changes and antibiotics concentration.
\(A_{0}+\frac{B_{0}}{A}=\frac{1}{\Delta N}\)

Then we want to develop a calibration with three point.

Figure 5: Experimental results of calibration

The fitting result indicate that our modeling and normalization operation was exactly correct and effective. OUR CRAZY MIND HIT OUR MODEL, AND OUR MODEL EXACYLY HIT OUR RESULT! WE LOVE SCIENCE!

That means the Processed Glass worth the name of special artificial original film!

Modeling guide us choose the candidates

Processing Film

Film I

Film II

Film III

You can detect various of kinds of material in solution, as long as you follow our standard to make the engineered bacteria. See our future NDM improvement plan at Future NDM

There is one thing to be sure is that whatever bacteria you used, you need to build calibration ruler to measure the concentration quantificationally. According to our modeling in calibration we need to confirm three constants 'A0', 'B0', 'n'.

\(A_{0}+\frac{B_{0}}{A}=\frac{1}{\Delta N}\)

That means we need to test at least three point to confirm the formula.

Establishement of the Calibration Ruler

To build a calibration ruler between chloramphenicol concentration and

interference fringes pattern.

In this case we use our E. coli BL21 with T7-OprF (BBa_K1593210), a type of bacteria in the best perfomance during genetically modification.(See our modification at Results-Permeability Improvement). According to our experiment results, we found the E. coli BL21 with T7-OprF (BBa_K1593210) was more sensitive to chloramphenicol.

And according to adhesion pre-experiment we chose the concentration range with 0.5ug/ml~5ug/ml in the experiment.

The changes of fringe number and test concentration shown below(take three pictures in a row with the time interval 20s),

Time(s)	5.0ug/ml(Fringe number)	1.0ug/ml(Fringe number)	0.5ug/ml(Fringe number)
0	42/43	51/49/52	44/43
20	-	50/51/50	-
40	-	50/46	-
60	-	48/48/50	45
80	-	48/49/49	-
100	-	51/53/49	45
120	42/43	-	-
140	-	51/48/52	-
160	-	-	41
180	-	48/52/54	-
200	48	55/47/49	43/43
220	43/47	51/52	45
240	48/46	53/51	44
starting average	42.5	50.5	43.5
ending average	46.4	51.75	44.2
ΔN	3.9	1.25	0.7

Then we can fit this data with calibration formula, assume n=3(through trying different value of n get the best value of n) then get the simplified formula:

\(A_{0}+\frac{B_{0}}{A}=\frac{1}{\Delta N}\)

Then the fitting result illstruates as,

And we can get the value of unknown constants:

constant	value
A0	0.1339
B0	0.6513

What an amazing result! The fitting result indicated that our modeling and normalization operation was exactly correct and effective. OUR CRAZY MIND HIT OUR MODEL, AND OUR MODEL EXACYLY HIT OUR RESULT! WE LOVE SCIENCE!

Reverification of Calibration

There may some arguement on our accuracy on calibration. So we need to conduct another experiment to reverficate our calibration ruler.

In order to test whether the calibration is correct and to confirm the coefficients, we choose two concentration as check point.

And use SPRING to measure its concentration. The real concentration and experiment results shown in table:

time(s)	1.8ug/ml(Fringe number)	3.2ug/ml(Fringe number)
0	78/80	77/76/77
20	77/77	79/77
40	78	75/72
60	79	74
80	81	75/75
100	75	72/73
120	80	74/74
140	83/82	71/75
160	81/79	72/76
180	82/82	75/74
200	82/81	75
220	80/82	74/74/75
240	81/80	74/75/74
starting average	79	77.2
ending average	81	74.3
ΔN	2.0	2.9
experiment concentration	1.80ug/ml	3.09ug/ml

The result showed that the experiment concentration is EXTREMELY CLOSE to the real concentration, which means OUR NDM IS ABLE TO TELL ANTIBIOTIC CONCENTRATION IN VERY ACCURATE WAY.

By the way, DO NOT FORGET, operating our NDM only cost you 100 SECONDS.

Yes, such fast and accurate hardware detecting antibiotics, that is our NDM, ONLY OUR NDM.

Note: To get the program of our calibration analysis, please visit Software. To know more information on how we analyzed our results, please visit Modeling: Calibration. To see our final measurement report with theoretical analysis, please visit Measurement.

Establishement of the Calibration Ruler
Reverification of Calibration

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-    <li><a href="https://2015.igem.org/Team:USTC/Tutorials" class="waves-effect waves-light">Tutorials</a></li>
+  <li><a href="https://2015.igem.org/Team:USTC/Measurement" class="waves-effect waves-light">Measurement</a></li>
-    <li><a href="https://2015.igem.org/Team:USTC/Notebook" class="waves-effect waves-light">NoteBook</a></li>
+  <li><a href="https://2015.igem.org/Team:USTC/Software" class="waves-effect waves-light">Software</a></li>
-    <li><a href="https://2015.igem.org/Team:USTC/Safety" class="waves-effect waves-light">Safety</a></li>
+  <li><a href="https://2015.igem.org/Team:USTC/Parts" class="waves-effect waves-light">Parts</a></li>
-    <li><a href="https://2015.igem.org/Team:USTC/Practices" class="waves-effect waves-light">Policy&Practices</a></li>
+  <li><a href="https://2015.igem.org/Team:USTC/Tutorials" class="waves-effect waves-light">Tutorials</a></li>
-    <li><a href="https://2015.igem.org/Team:USTC/Collaborations" class="waves-effect waves-light">Collaborations</a></li>
+  <li><a href="https://2015.igem.org/Team:USTC/Notebook" class="waves-effect waves-light">NoteBook</a></li>
-    <li><a href="https://2015.igem.org/Team:USTC/Team" class="waves-effect waves-light">Team</a></li>
+  <li><a href="https://2015.igem.org/Team:USTC/Safety" class="waves-effect waves-light">Safety</a></li>
-    <li><a href="https://2015.igem.org/Team:USTC/Attributions" class="waves-effect waves-light">Attributions</a></li>
+  <li><a href="https://2015.igem.org/Team:USTC/Practices" class="waves-effect waves-light">Policy&Practices</a></li>
+  <li><a href="https://2015.igem.org/Team:USTC/Team" class="waves-effect waves-light">Team</a></li>
+  <li><a href="https://2015.igem.org/Team:USTC/Collaborations" class="waves-effect waves-light">Collaborations</a></li>
+  <li><a href="https://2015.igem.org/Team:USTC/Attributions" class="waves-effect waves-light">Attributions</a></li>
 </ul>
@@ Line 43: / Line 53: @@
          <div id="Overview" class="row">
              <div class="card hoverable">
-                 <div class="col s12">
+                 <div class="col s12 m9">
                      <div class="card-content">
                          <p>That is so amazing! We finally made NDM this year! The results page will deliver the fresh
@@ Line 59: / Line 69: @@
                                  results.</p>
                              </li>
-                             <li><p>Antibiotics Concentration Detection Demo: This section includes our initial test on
+                             <li><p>Film Determination: This section introduced how we finally determinated our film,
-                                 our system, how we define our final methods and how we establshed protocols for
+                                 from plastic film to our final candidate. See how difficult and interesting it is!</p>
-                                users.</p>
                              </li>
-                             <li><p>Calibration</p>
+                             <li><p>Calibration: This is our final step demonstrating the feasibility of NDM. See how
+                                perfect our project is!</p>
                              </li>
                          </ul>
-                        <p>Want to get them now? Click their title directly!</p>
                      </div>
@@ Line 74: / Line 83: @@
          </div>
+         <div id="Permeability_Improvement" class="row">
-         <div id="Chemotaxis_Modification" class="row">
              <div class="card hoverable">
                  <div class="row">
-                     <div class="col s12">
+                     <div class="col s12 m9">
                          <div class="card-content">
-                             <p><strong>Growth characteristics</strong><br>We detected OD600(nm) to identify the effect
+                             <h4 id="Extraction_of_Permeability_Improvement_Fragements" class="scrollspy">Extraction of
-                                of cheZ expression on <em>E. coli</em> through time.<br><img
+                                Permeability Improvement Fragements</h4>
-                                        src="https://static.igem.org/mediawiki/2015/d/d6/Ustc-growth.png" alt="图片名称"></p>
-                             <p>The exogenous protein expression affects the growth of bacteria, which was indicated by
+                             <p>We successfully get
-                                 slight slower grow-up in <em>E. coli</em> containing cheZ plasmid. In total, cheZ
+                                gene fragements that are used for improving bacterial permeability, which including,
-                                 doesn't have obvious negative impact on bacteria grown. </p>
+                            </p>
+                            <ul>
+                                <li>SCVE, a viroporin originally from SARS virus, is synthesized by Sangon Co. ctd.</li>
+                            </ul>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/1/1d/Ustc-scve.jpeg">
+                              <figcaption>
+                              Figure 1: Extraction of SCVE fragement
+                              </figcaption>
+                           </figure>
+                            <ul>
+                                <li>OprF, a bigger porin compared with OmpF in <em>E. coli</em>, is extracted from <em>P.aeruginosa，
+                                    PAO1</em> genome by PCR.
+                                </li>
+                            </ul>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/6/6d/Ustc-t7oprf_lac-chez.jpeg">
+                              <figcaption>
+                              Figure 2: Extraction of OprF fragement
+                              </figcaption>
+                           </figure>
+                            <ul>
+                                <li><p>T7 is a strong promoter which we got form Parts Registry. T7 will be used to
+                                    trigger the expression of both OprF and SCVE.<br>
+                              <figure>
+                                <img src="https://static.igem.org/mediawiki/2015/7/7a/Ustc-T7.jpeg">
+                                <figcaption>
+                                Figure 3: T7 extraction from parts
+                                </figcaption>
+                              </figure>
+                                </li>
+                                <li><p>Cas9 is got from 2015 Part Distribution.</p>
+                                </li>
+                            </ul>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/1/1c/USTC-Cas9.png">
+                              <figcaption>
+                              Figure 4: Extraction of Cas9 from parts
+                              </figcaption>
+                           </figure>
+                            <ul>
+                                <li>gRNA-AcrB and gRNA-EmrE, these two fragments are for silencing bacterial
+                                    transmembrane protein AcrB and EmrE to strongly block drug efflux system. These two
+                                    parts are synthesized by Sangon Biotech Company.
+                                </li>
+                            </ul>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/5/54/USTC-gRNA_extraction.png">
+                              <figcaption>
+                              Figure 5: Extraction of gRNA-AceB and gRNA-EmrE, two fragments are synthesized by Sangon Biotech Company
+                              </figcaption>
+                           </figure>
+                            <h4 id="Construction_of_Permeability_Improving_Plasmid" class="scrollspy">Construction of
+                                Permeability Improving Plasmid</h4>
+                            <ul>
+                                <li>Construction of plasimid with T7-SCVE (<a
+                                        href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>) . We finally
+                                    ligated the T7 with SCVE and got T7-SCVE (<a
+                                            href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>).
+                                </li>
+                            </ul>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/e/ed/Ustc-scve_jp_t7.jpeg">
+                              <figcaption>
+                              Figure 6: Construction of T7-SCVE ([BBa_K1593667](http://parts.igem.org/Part:BBa_K1593667))
+                              </figcaption>
+                           </figure>
+                            <ul>
+                                <li>Construction of plasimid containing T7-OprF (<a
+                                        href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>). This is the
+                                    result of T7 with OprF.
+                                </li>
+                            </ul>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/6/6d/Ustc-t7oprf_lac-chez.jpeg">
+                              <figcaption>
+                              Figure 7: Construction of T7-OprF ([BBa_K1593210](http://parts.igem.org/Part:BBa_K1593210))
+                              </figcaption>
+                           </figure>
+                            <h4 id="Growth_Characterization" class="scrollspy">Growth Characterization</h4>
+                            <p>We firstly characterized the growth rate of genetically modified CACCI along with wild
+                                type BL21 using optical density at 600 nm, which is called OD 600, in order to
+                                demonstrate that the overexpression of porin(OprF with T7,<a href="http://parts.igem.org/Part:BBa_K1593209">BBa_K1593209</a>)
+                                or viroporin(SCVE with T7, (<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
+                                won't severely affect the growth of bacteria, at least it won't significantly inhibit
+                                bacteria growth and become a kill switch.</p>
+                            <p>All bacteria are cultured previously in LB medium about 12 h. And then the measurement of
+                                bacteria growth through time began at 8 A.M. the other day.</p>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/d/d8/Ustc-growth2.png">
+                              <figcaption>
+                              Figure 8: Bacteria growth characterization
+                              </figcaption>
+                           </figure>
+                            <p>As the images above illustrated, we found <em>E. coli</em> BL21 wild type has the best
+                                growth characteristics with a maximal OD600=3.427. Whereas <em>E. coli</em> BL21 with
+                                OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) has a
+                                relatively low optical density as its OD600 after 12 h is 2.808 compared to the
+                                wildtype. In the same way, characterization of <em>E. coli</em> BL21 with SCVE, <a
+                                        href="http://parts.igem.org/Part:BBa_K1593667">http://parts.igem.org/Part:BBa_K1593667</a>
+                                showed a slightly smaller OD600, as its finally turned to 2.835. <em>E. coli</em> BL21
+                                with T7-OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) and
+                                <em>E. coli</em> BL21 with T7-SCVE, (<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
+                                are showing 19% decreased maximal cell density and 18.5% decreased maximal cell density
+                                respectively in comparison with wild type.</p>
+                            <p>Consequently, the result implies that the genetical modification on bacterial
+                                permeability, at least the overexpression of OprF and SCVE with a strong promoter T7,
+                                won't significantly influence the bacterial growth. Thus, genetically modified bacteria
+                                can be used in the following experiments.</p>
+                            <p>Later on, we will characterize the basic permeability capability of <em>E. coli</em> BL21
+                                with T7-OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) and
+                                <em>E. coli</em> BL21 with T7-SCVE, (<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>).
+                            </p>
+                            <h4 id="ONPG_Assay" class="scrollspy">ONPG Assay</h4>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/5/54/ONPG-ustc-sche.png">
+                              <figcaption>
+                              Figure 9: ONPG reaction
+                              </figcaption>
+                           </figure>
+                            <p>ONPG, that is the abbreviation of ortho-Nitrophenyl-β-galactoside. ONPG is a colorimetric
+                                and spectrophotometric substrate, previously used for detecting β-galactosidase
+                                activity. ONPG is actually colorless at normal situation, however if it triggers the
+                                degradation reaction catalyzed by β-galactosidase, we will get galactose and
+                                ortho-nitrophenol. Surprisingly, the compound ortho-nitrophenol has a yellow color,
+                                which means we are able to detect the chemical reaction through OD detection. Besides,
+                                owing to the existence of β-galactosidase in bacteria, a more intense optical density we
+                                detected, that means there are more ONPG uptaked by bacteria. Therefore, using ONPG we
+                                 are able to distinguish the permeability capability of different bacteria.</p>
+                            <p>Here's our result.</p>
+                            <p>Note: The wavelength for detecting the bacterial concentration and absortion of ONPG are
+                                respectively 600nm and 406nm.</p>
+                            <p>First, we detected the bacterial solutions to control the initial concentrations. </p>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/3/32/Ustc-board.png">
+                              <figcaption>
+                              Figure 10: ONPG assay schematic show
+                              </figcaption>
+                           </figure>
+                            <p>The OD values for <em>E.coli </em>BL21, <em>E.coli </em>BL21 with T7-OprF (<a
+                                    href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) , <em>E.coli</em>
+                                BL21 with T7-SCVE(<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
+                                are respectively 2.856,1.806 and 1.889. Based on these, we know that the concentrations
+                                of these three bacteria are close.</p>
+                            <p>Then we add ONPG to the bacterial solutions and detect the OD.</p>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/5/52/Ustc-onpg88.png">
+                              <figcaption>
+                              Figure 11: ONPG assay, absorption OD406
+                              </figcaption>
+                           </figure>
+                            <p>From this figure, we can see a rising trend in all bacterial solutions. The OD values for
+                                <em>E.coli</em> BL21 with T7-OprF(<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>)
+                                and <em>E.coli </em>BL21 with T7-SCVE(<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
+                                are respectively 0.2520 and 0.2588,both are higher than <em>E.coli</em> BL21,the wild
+                                type. We can infer that the absorbtion of modified bacteria is higher than the wild
+                                type.</p>
+                            <p>There is a more visual figure for comparing the absorbtions. We made a zero calibration
+                                using the OD value of wild type as the zero level.</p>
+                            <figure>
+                              <img src="https://static.igem.org/mediawiki/2015/0/00/Ustc-onpg55.png">
+                              <figcaption>
+                              Figure 12: ONPG assay, absorption OD406 in average
+                              </figcaption>
+                           </figure>
+                            <p>In general, the OD values of both two modified bacteria are rising. For<em> E.coli</em>
+                                 BL21 with T7-OprF(<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>),
+                                the rising is relatively smooth. For <em>E.coli</em> BL21 with T7-SCVE(<a
+                                        href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>),the value goes
+                                down first, then goes up .And the <em>E.coli</em> BL21 with T7-SCVE(<a
+                                        href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>) has a higher
+                                absorbtion than <em>E.coli </em>BL21 with T7-OprF(<a
+                                        href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>).</p>
+                            <h4 id="NPN_Assay" class="scrollspy">NPN Assay</h4>
+                            <figure><img
+                                    src="https://static.igem.org/mediawiki/2015/1/16/Ustc-npn.gif"
+                                    alt="Figure 13: Schematic Structure of N-Phenyl-1-naphthylamine, NPN"><figcaption>Figure 13: Schematic Structure of N-Phenyl-1-naphthylamine, NPN</figcaption></figure>
+                            <p>N-Phenyl-1-naphthylamine(NPN),C16H13N, CAS:90-30-2, FW: 219.29, is a nonpolar probe,
+                                whose fluorescence signal is relatively strong in a phospholipid environment, but weak
+                                in aqueous surroundings. Consequently, using this feature, we are able to characterize
+                                bacteria permeability by measuring the fluorescence intensity of the bacteria solution.
+                                When NPN molecules are absorbed by bacteria, we will observe a significantly rising
+                                fluorescence intensity. The more fluorescence intensity we got, the more permeable the
+                                bacteria are. Here are what we got from NPN uptake assay and the conclusion on
+                                genetically engineered bacteria permeability improvement. </p>
+                            <p>Here's our results, the first picture illustrate the total fluorescence intensity
+                                measurement by <a href="http://www.bmglabtech.com/en/products/clariostar/">BMG Labtech
+                                    CLARIOstar®</a>.</p>
+                            <p><em>Note</em>: The fluorescence intensity signal at excitation wavelength range is 305nm
+                                to 335nm and emission wavelength range is 370nm to 410nm, which is recommended by
+                                previous research. </p>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/e/e5/Ustc-npn1.png"
+                                    alt="Figure 14: NPN uptake result-1"><figcaption>Figure 14: NPN uptake result-1</figcaption></figure>
+                            <p>The total fluorescence intensity of <em>E. coli</em> BL21 wild type is 126451. On the
+                                other hand, the fluorescence intensity in <em>E. coli</em> BL21 with T7-OprF (<a
+                                        href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) and <em>E.
+                                    coli</em> BL21 with T7-SCVE, (<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
+                                are respectively 207322 and 151820, which are significantly larger than the wildtype
+                                assay.</p>
+                            <p>Though we have already got the exact data on NPN uptake, we still need to revise its
+                                effectiveness because of the different bacteria concentration of <em>E. coli</em> BL21
+                                wild type, <em>E. coli</em> BL21 with T7-OprF (<a
+                                        href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) and <em>E.
+                                    coli</em> BL21 with T7-SCVE, (<a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>).
+                                Here is the exact bacteria concentration data, based on OD600nm,</p>
+                            <table class="striped">
+                                <thead>
+                                <tr>
+                                    <th>Strain</th>
+                                    <th>OD600nm</th>
+                                </tr>
+                                </thead>
+                                <tbody>
+                                <tr>
+                                    <td><em>E. coli</em> BL21 wild type</td>
+                                    <td>3.663</td>
+                                </tr>
+                                <tr>
+                                    <td><em>E. coli</em> BL21 with T7-OprF (<a
+                                            href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>)
+                                    </td>
+                                    <td>2.431</td>
+                                </tr>
+                                <tr>
+                                    <td><em>E. coli</em> BL21 with T7-SCVE, (<a
+                                            href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>)
+                                    </td>
+                                    <td>2.441</td>
+                                </tr>
+                                </tbody>
+                            </table>
+                            <p>After correcting these data, we are able to get bacteria absorption uptake capability in
+                                average, which is a more accurate value to characterize bacterial permeability
+                                property.</p>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/1/1a/Ustc-npn2.png"
+                                    alt="Figure 15: NPN uptake result in average "><figcaption>Figure 15: NPN uptake result in average</figcaption></figure>
+                            <p>The fluorescence intensity in average of <em>E. coli</em> BL21 wild type is 34521.16. On
+                                the contrary, the fluorescence intensity revised by bacterial concentration in <em>E.
+                                    coli</em> BL21 with T7-OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>)
+                                and <em>E. coli</em> BL21 with T7-SCVE, (<a
+                                        href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>) are
+                                respectively 85282.60 and 62195.8, which are relatively 2.5 folds and approximately 2
+                                folds to <em>E. coli</em> wild type, that is a significantly improvement compared to
+                                wild type.</p>
+                            <p>Consequently, through NPN uptake assay, we are able to conclude that the small molecule
+                                uptake capability of bacteria improved after our genetical modification. Thus <em>E.
+                                    coli</em> BL21 with T7-OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>)
+                                and <em>E. coli</em> BL21 with T7-SCVE, (<a
+                                        href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a>) are both able
+                                to use as the candidate bacteria strains for NDM measurement.</p>
+                            <h4 id="bibliography" class="scrollspy">Bibliography</h4>
+                            <ol>
+                                <li><p><em>T. Mattila-Sandholm et al.</em> Fluorometric assessment of Gram-negative
+                                    bacterial permeabilization. <strong>Journal of Applied Microbiology</strong> 2000,
+, 213–219</p>
+                                </li>
+                                <li><p><em>Scott Banta et al.</em> Genetic Manipulation of Outer Membrane Permeability:
+                                    Generating Porous Heterogeneous Catalyst Analogs in <em>Escherichia coli</em>
+                                    dx.doi.org/10.1021/sb400202s <strong>ACS Synth. Biol.</strong> 2014, 3, 848−854</p>
+                                </li>
+                            </ol>
-                            <p><strong>SDS-PAGE</strong><br>After induced by IPTG in 1mM about 4 h, cheZ was found in
+                        </div>
-                                 SDS-PAGE gel, compared to the wild type (Top10) and bacteria without IPTG induction.
+                    </div>
-                                 Through this result, we concluded that cheZ has been successfully expressed with the
+                    <div class="col hide-on-small-only m3">
-                                 IPTG induction.<br><img
+                        <div class="toc-wrapper pinned">
-                                        src="https://static.igem.org/mediawiki/2015/7/72/Ustc-chez_expression.png" alt="图片名称"><br>See
+                            <ul class="section table-of-contents">
-                                 protocol in <a href="http://tower.im">Protocols: SDS-PAGE</a></p>
+                                 <li>
+                                    <a href="#Extraction_of_Permeability_Improvement_Fragements">Extraction of
+                                        Permeability Improvement Fragement</a>
+                                 </li>
+                                 <div class="divider"></div>
+                                <li>
+                                    <a href="#Construction_of_Permeability_Improving_Plasmid">Construction of
+                                        Permeability Improving Plasmid</a>
+                                </li>
+                                <div class="divider"></div>
+                                <li>
+                                    <a href="#Growth_Characterization">Growth Characterization</a>
+                                </li>
+                                <div class="divider"></div>
+                                <li>
+                                    <a href="#ONPG_Assay">ONPG Assay</a>
+                                    <div class="divider"></div>
+                                 <li>
+                                    <a href="#NPN_Assay">NPN Assay</a>
+                                    <div class="divider"></div>
+                                <li>
+                                    <a href="#bibliography">Bibliography</a>
+                                </li>
+                            </ul>
                          </div>
                      </div>
@@ Line 100: / Line 426: @@
          </div>
-         <div id="Permeability_Improvement" class="row">
+         <div id="Chemotaxis_Modification_and_ROSE" class="row">
              <div class="card hoverable">
                  <div class="row">
-                     <div class="col s12">
+                     <div class="col s12 m9">
                          <div class="card-content">
-                             <h3>Extraction of gene fragements</h3>
+                             <h4 id="Genetic_Fragments_Extraction" class="scrollspy">Genetic Fragments Extraction</h4>
-                            <p>We successfully get gene fragements that are used for bacterial permeability improvement,
-                                which including, </p>
                              <ul>
-                                 <li><p>micF and SoxS, these two antibiotic substance responding promoters were extracted
+                                 <li>micF and SoxS, these two antibiotic substance responding promoters were extracted
-                                     from <em>E. coli Top 10(K-12 strain)</em> by conducting polymerase chain reaction on
+                                     from E. coli Top 10(K-12 strain) by conducting polymerase chain reaction on its
-                                     its genome.<br><img src="https://static.igem.org/mediawiki/2015/2/2b/MicF_and_SoxS.png"
+                                     genome.
-                                                        alt="图片名称"></p>
                                  </li>
-                                 <li><p>SCVE, a viroporin originally from SARS virus, is synthesized from Sangon Co. ctd.<br><img
+                            </ul>
-                                         src="https://static.igem.org/mediawiki/2015/1/12/USTC-SCVE.png" alt="图片名称"></p>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/7/7e/Ustc-micf_soxs_xh.jpeg"
+                                    alt="Figure 1: fragements extraction of micF and SoxS"><figcaption>Figure 1: fragements extraction of micF and SoxS</figcaption></figure>
+                            <ul>
+                                 <li>cheZ, this is the chemotaxis modified fragment.</li>
+                            </ul>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/8/8c/Ustc-chez.jpeg"
+                                    alt="Figure 2: fragements extraction of cheZ"><figcaption>Figure 2: fragements extraction of cheZ</figcaption></figure>
+                            <ul>
+                                <li>BBa_R0010, this is promoter of lac, which triggers the expression of cheZ.</li>
+                            </ul>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/3/34/Ustc-oprf_ep_r0010.jpeg"
+                                    alt="[Figure 3: fragment extraction of OprF and Lac](http://parts.igem.org/Part:BBa_K1593997)">
+                            <figcaption></figcaption>[Figure 3: fragment extraction of OprF and Lac](http://parts.igem.org/Part:BBa_K1593997)</figure>
+                            <p><strong>Construction of plasimid with lac-cheZ <a
+                                    href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a></strong></p>
+                            <p>This is our final construction of lac-cheZ <a
+                                    href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a></p>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/6/6d/Ustc-t7oprf_lac-chez.jpeg"
+                                    alt="Figure 4: plasmid construction of lac-cheZ [BBa_K1593997](http://parts.igem.org/Part:BBa_K1593997) "><figcaption>Figure 4: plasmid construction of lac-cheZ [BBa_K1593997](http://parts.igem.org/Part:BBa_K1593997)</figcaption>
+                            </figure>
+                            <h4 id="Growth_characteristics" class="scrollspy">Growth characteristics</h4>
+                            <p>We detected OD600(nm) to identify the effect
+                                of cheZ (<a href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a>) expressed
+                                on <em>E. coli</em> BL21 with time induced by IPTG or not.</p>
+                            <figure><img src="https://static.igem.org/mediawiki/2015/d/d6/Ustc-growth.png"
+                                    alt="Figure 5: Growth characteristics of BL21 WT with lac-cheZ [BBa_K1593997](http://parts.igem.org/Part:BBa_K1593997) induced by IPTG or not"><figcaption>Figure 5: Growth characteristics of BL21 WT with lac-cheZ [BBa_K1593997](http://parts.igem.org/Part:BBa_K1593997) induced by IPTG or not</figcaption>
+                            </figure>
+                            <p>As the images above illustrated, we found <em>E. coli</em> BL21 wild type has the best
+                                growth characteristics with a maximal OD600=2.6. Whereas <em>E. coli</em> BL21 with cheZ
+                                (<a href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a>) induced by ITPG has
+                                a relatively low optical density as its OD600 after 9 h is 2.3 compared to the wildtype.
+                                In the same way, characterization of <em>E. coli</em> BL21 with cheZ (<a
+                                        href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a>) not induced by
+                                IPTG showed a slightly smaller OD600, as its finally turned to 2.1. The exogenous
+                                protein expression affects the growth of bacteria, which was indicated by slight slower
+                                grow-up in <em>E. coli</em> containing cheZ plasmid. In total, cheZ doesn't have obvious
+                                negative impact on bacteria grown. </p>
+                            <h4 id="SDS-PAGE" class="scrollspy">SDS-PAGE</h4>
+                            <p>After induced by IPTG in 1mM about 4 h, cheZ was found in
+                                SDS-PAGE gel, compared to the wild type (Top10) and bacteria without IPTG induction.
+                                Through this result, we concluded that cheZ has been successfully expressed with the
+                                IPTG induction.</p>
+                            <figure><img
+                                         src="https://static.igem.org/mediawiki/2015/7/72/Ustc-chez_expression.png"
+                                        alt="Figure 6: characterization of cheZ expression by SDS-PAGE"><figcaption>Figure 6: characterization of cheZ expression by SDS-PAGE</figcaption></figure>
+                            <p>See protocol
+                                in <a href="https://2015.igem.org/Team:USTC/Notebook#Protocols">Protocols</a></p>
+                        </div>
+                    </div>
+                    <div class="col hide-on-small-only m3">
+                        <div class="toc-wrapper pinned">
+                            <ul class="section table-of-contents">
+                                <li>
+                                    <a href="#Genetic_Fragments_Extraction">Genetic Fragments Extraction</a>
                                  </li>
-                                 <li>OprF, a bigger porin compared with OmpF in <em>E. coli</em>, is extracted from <em>P.aeruginosa，
+                                 <div class="divider"></div>
-                                     PAO1</em> genome by PCR.<br><img
+                                <li>
-                                        src="https://static.igem.org/mediawiki/2015/6/66/USTC-OprF.png" alt="图片名称"></li>
+                                     <a href="#Growth_characteristics">Growth characteristics</a>
-                                 <li>CheZ, a chemotaxis related protein is also extracted from <em>P.aeruginosa，
+                                </li>
-                                     PAO1</em> genome by PCR.
+                                <div class="divider"></div>
+                                 <li>
+                                     <a href="#SDS-PAGE">SDS-PAGE</a>
                                  </li>
-                                <li>Cas9, from 2015 part registry.<br><img
-                                        src="https://static.igem.org/mediawiki/2015/1/1c/USTC-Cas9.png" alt="图片名称"></li>
-                                <li>gRNA-AcrB and gRNA-EmrE, these two fragments is for genetically modifed bacterial
-                                    transmembrane protein AcrB and EmrE to strongly block drug efflux system.<br><img
-                                            src="https://static.igem.org/mediawiki/2015/5/54/USTC-gRNA_extraction.png"
-                                            alt="图片名称"></li>
                              </ul>
                          </div>
@@ Line 136: / Line 515: @@
              </div>
          </div>
          <div id="Adhesion_Assay" class="row">
              <div class="card hoverable">
-                 <div class="col s12">
+                 <div class="col s12 m9">
                      <div class="card-content">
-                         <h3 id="characterization-of-optimal-conditions-on-polylysine-pll-coated-assay">Characterization
+                         <h4 id="characterization-of-optimal-conditions-on-polylysine-pll-coated-assay"
-                             of Optimal Conditions on Polylysine(PLL) Coated Assay</h3>
+                            class="scrollspy">
+                             Characterization of Optimal Conditions on Polylysine(PLL) Coated Assay</h4>
                          <p>To see the details on the mechanism of polylysine adhesion, please refer to <a
-                                 href="http://tower.im">Adhesion: Polylysine</a>.</p>
+                                 href="https://2015.igem.org/Team:USTC/Description#CACCI">Project-CACCI</a>.</p>
-                         <p>To get the final PLL-coated protocol, check <a href="http://tower.im">Protocols: PLL Coated
+                         <p>To get the final PLL-coated protocol, check <a href="https://2015.igem.org/Team:USTC/Notebook#Protocols">Protocols</a> for further information.</p>
-                            Assay</a> for further information.</p>
-                         <p>Our original characterization of optimal conditions on polylysine(PLL)-coated assay requires
+                         <p>Our original characterization of optimal conditions on polylysine(PLL)-coated assay
-                             several factors to get, including:</p>
+                             requires several factors to get, including:</p>
                          <ol>
-                             <li>Best bacterial developmental interval along with recommended dilution conditions.</li>
+                             <li>Best bacterial developmental interval along with recommended dilution conditions.
+                            </li>
                              <li>Best PLL-Coated Concentration and Pre-treatment Time.</li>
-                             <li>Best PLL-Coated Time, given full consideration to the completion of bacterial adhesion
+                             <li>Best PLL-Coated Time, given full consideration to the completion of bacterial
-                                 time.
+                                 adhesion time.
                              </li>
-                             <li>Best Measurement Interval, which illustrates the possible time for bacterial response on
+                             <li>Best Measurement Interval, which illustrates the possible time for bacterial
-                                 antibiotics pressure.
+                                 response on antibiotics pressure.
                              </li>
                              <li>Possible Determination Interval, which refers to antibiotics response interval.</li>
@@ Line 166: / Line 547: @@
                              antibiotics using our CACCI and SPRING.</p>
-                         <p><strong>Best Bacterial Developmental Interval Along with Recommended Dilution
+                         <h4 id="Best_Bacterial_Developmental_Interval_Along_with_Recommended_Dilution_Conditions"
-                             Conditions</strong></p>
+                            class="scrollspy">Best Bacterial Developmental Interval Along with Recommended Dilution
+                             Conditions</h4>
-                         <p>According to experience on <em>E. coli</em> development based on OD detector. We concluded
+                         <p>According to experience on <em>E. coli</em> development based on OD detector. We
-                             that bacteria exploding during <strong>Logarithmic Period(OD:535nm about 0.4~0.5)</strong>
+                             concluded that bacteria exploding during <strong>Logarithmic Period(OD:535nm about
-                            are at the most energetic moments with proper bacterial density. Consequently, we highly
+.4~0.5)</strong> are at the most energetic moments with proper bacterial density.
-                            recommend user to culture bacteria at Logarithmic Period and then <strong>dilute bacterial
+                            Consequently, we highly recommend user to culture bacteria at Logarithmic Period and
-                                solution about twenty times to fifty times.</strong></p>
+                            then <strong>dilute bacterial solution about twenty times to fifty times.</strong></p>
-                         <p><strong>Best PLL-Coated Concentration and Pre-treatment Time.</strong></p>
+                         <h4 id="Best_PLL-Coated_Concentration_and_Pre-treatment_Time" class="scrollspy">Best PLL-Coated
+                            Concentration and Pre-treatment Time.</h4>
-                         <p>Best PLL-Coated Conditions, which include PLL-Pretreatment time, PLL-Coated concentration and
+                         <p>Best PLL-Coated Conditions, which include PLL-Pretreatment time, PLL-Coated concentration
-                             PLL-Coated time are measured for best adhesive condictions for bacterial treatment.</p>
+                             and PLL-Coated time are measured for best adhesive condictions for bacterial
+                            treatment.</p>
-                         <p>As for PLL-Pretreatment time, we adopt traditional pretreatment time, incubating about 12~16
+                         <p>As for PLL-Pretreatment time, we adopt traditional pretreatment time, incubating about
-                            h, for recommendation, which is originally used for neurons coated on neural science
+~16 h, for recommendation, which is originally used for neurons coated on neural
-                             research. And when it comes to PLL-coated concentration, experience on neurons adhesion is
+                             science research. And when it comes to PLL-coated concentration, experience on neurons
-                             also precious. According to previous research, PLL concentration in the interval of 20 ug/mL
+                             adhesion is also precious. According to previous research, PLL concentration in the
-                            to 100 ug/mL is quite effective for bacterial adhesion.</p>
+                            interval of 20 ug/mL to 100 ug/mL is quite effective for bacterial adhesion.</p>
-                         <p><strong>Adhesion Assay with PLL treatment</strong><br>Here we deliver the PLL treatment
+                         <h4 id="Adhesion_Assay_with_PLL_treatment" class="scrollspy">Adhesion Assay with PLL
-                             results comparing with no PLL treatment assay. During pre-experiment adhesion assay, PAO1, a
+                             treatment</h4>
-                            strain of <em>Pseudomonas aeruginosa</em>, is used for adhesion effects. </p>
-                         <p>The picture below showed the amount of bacteria(PAO1) under microscope without PLL treatment,
+                         <p>Here we deliver the PLL treatment
-                             which is abbreviated as PLL(-). To observe capability of bacterial adhesion, we wash
+                             results comparing with no PLL treatment assay. During pre-experiment adhesion assay,
-                             observed place with PBS:</p>
+                             PAO1, a strain of <em>Pseudomonas aeruginosa</em>, is used for adhesion effects. </p>
-                         <p><img src="https://static.igem.org/mediawiki/2015/c/c3/Control_-1.png"
+                         <p>The picture below showed the amount of bacteria(PAO1) under microscope without PLL
-                                alt="Control Assay-Without PLL treatment"></p>
+                            treatment, which is abbreviated as PLL(-). To observe capability of bacterial adhesion,
+                            we wash observed place with PBS:</p>
-                         <p>After elution, a significant decline in the number of bacteria is recorded. As a matter of
+                         <figure><img src="https://static.igem.org/mediawiki/2015/c/c3/Control_-1.png"
-                            fact, there is no bacteria in observed field after PBS washing. </p>
+                                alt="Figure 1: Control Assay-Without PLL treatment"><figcaption>Figure 1: Control Assay-Without PLL treatment</figcaption></figure>
-                         <p><em>To get the algorithm of bacterial counting, please refer to <a href="http://tower.im">Modeling:
+                        <p>After elution, a significant decline in the number of bacteria is recorded. As a matter
-                            Bacterial number and movement analysis</a></em>.</p>
+                            of fact, there is no bacteria in observed field after PBS washing. </p>
+                         <p><em>To get the algorithm of bacterial counting, please refer to <a
+                                href="https://2015.igem.org/Team:USTC/Modeling#Adhesion-Dynamics">Modeling: Adhesion Dynamics</a></em>.
+                        </p>
-                         <p><img src="https://static.igem.org/mediawiki/2015/e/e9/PAO1-PLL--.png"
+                         <figure><img src="https://static.igem.org/mediawiki/2015/e/e9/PAO1-PLL--.png"
-                                 alt="Control Assay-Without PLL treatment2"></p>
+                                 alt="Figure 2: Control Assay-Without PLL treatment2"><figcaption>Figure 2: Control Assay-Without PLL treatment2</figcaption></figure>
                          <p>As for treatment with PLL in 20 ug/mL, which is abbreviated as PLL(+,20), the impressive
-                             adhesion effect is observed. To ensure the exact effect, bacteria observing field is washed
+                             adhesion effect is observed. To ensure the exact effect, bacteria observing field is
-                             twice by PBS.</p>
+                             washed twice by PBS.</p>
-                         <p><img src="https://static.igem.org/mediawiki/2015/9/96/Adhesion_Assay.png"
+                         <figure><img src="https://static.igem.org/mediawiki/2015/9/96/Adhesion_Assay.png"
-                                 alt="Adhesion Assay-With PLL treatment in 20 ug/mL"></p>
+                                 alt="Figure 3: Adhesion Assay-With PLL treatment in 20 ug/mL"><figcaption>Figure 3: Adhesion Assay-With PLL treatment in 20 ug/mL</figcaption></figure>
                          <p>The number of bacteria doesn't decline, and on the contrary, we see slight increase of
-                             bacteria number. The possible reason is constant bacterial settlement during adhesion assay.
+                             bacteria number. The possible reason is constant bacterial settlement during adhesion
-                             After twice PBS wash, the number of bacteria is relatively stable as well. Consequently, we
+                             assay. After twice PBS wash, the number of bacteria is relatively stable as well.
-                            concluded PLL has significant adhesive ability on bacteria.</p>
+                            Consequently, we concluded PLL has significant adhesive ability on bacteria.</p>
-                         <p><img src="https://static.igem.org/mediawiki/2015/9/9e/PAO-Withoug.png"
+                         <figure><img src="https://static.igem.org/mediawiki/2015/9/9e/PAO-Withoug.png"
-                                 alt="Adhesion Assay-With PLL treatment in 20 ug/mL"></p>
+                                 alt="Figure 4: Adhesion Assay-With PLL treatment in 20 ug/mL"><figcaption>Figure 4: Adhesion Assay-With PLL treatment in 20 ug/mL</figcaption></figure>
-                         <p><strong>Best PLL-Coated Time</strong><br>Then, we tried to figure out the cohesive effect of
+                         <h4 id="Best_PLL-Coated_Time" class="scrollspy">Best PLL-Coated Time</h4>
-                             polylysine through time to get the best PLL-coated time. We gathered data just after PLL
-                             treatment(0s), after 1min and after 5min. And we also recorded bacteria number in the
+                        <p>Then, we tried to figure out the cohesive effect
-                             following 20 s. Here we provide the analysis of bacterial number variation after PLL
+                             of polylysine through time to get the best PLL-coated time. We gathered data just after
-                             treatment.We respectively use PAO1, a kind of <em>Pseudomonas aeruginosa</em> and HCB1, a
+                             PLL treatment(0s), after 1min and after 5min. And we also recorded bacteria number in
-                             kind of <em>E. coli</em> to handle the assay. PAO1 contains self-adhesive ability and HCB1
+                             the following 20 s. Here we provide the analysis of bacterial number variation after PLL
-                             has strong mobile ability. Using these genetically natural bacteria, we would conclude with
+                             treatment.We respectively use PAO1, a kind of <em>Pseudomonas aeruginosa</em> and HCB1,
-                             the effect of polylysine treatment.</p>
+                             a kind of <em>E. coli</em> to handle the assay. PAO1 contains self-adhesive ability and
+                             HCB1 has strong mobile ability. Using these genetically natural bacteria, we would
+                             conclude with the effect of polylysine treatment.</p>
                          <p><em>As for PAO1</em>, Without polylysine coated, bacteria have strong swimming ability.
-                             Because PAO1 is a kind of self-adhesive bacteria for experiment, thus we could see adhesive
+                             Because PAO1 is a kind of self-adhesive bacteria for experiment, thus we could see
-                             bacteria increasing during assay.</p>
+                             adhesive bacteria increasing during assay.</p>
-                         <p><img src="https://static.igem.org/mediawiki/2015/5/5c/Ustc-2015-pll90.png" alt="图片名称"></p>
+                         <figure><img src="https://static.igem.org/mediawiki/2015/5/5c/Ustc-2015-pll90.png"
+                                alt="Figure 5: no antibiotics bacterial number variation through time without PLL">
+                        <figcaption>Figure 5: no antibiotics bacterial number variation through time without PLL</figcaption></figure>
-                         <p>With 20 ug/mL polylysine treatement, bacterial adhesive effect due to strong electrostatic
+                         <p>With 20 ug/mL polylysine treatement, bacterial adhesive effect due to strong
-                             adhesion becomes stronger, and we, as well are able to observe the number of adherent
+                             electrostatic adhesion becomes stronger, and we, as well are able to observe the number
-                             bacteria gradually rising until reaching equilibrium. </p>
+                             of adherent bacteria gradually rising until reaching equilibrium. </p>
-                         <p><img src="https://static.igem.org/mediawiki/2015/e/ee/Developemnt.png" alt="图片名称"></p>
+                         <figure><img src="https://static.igem.org/mediawiki/2015/e/ee/Developemnt.png"
+                                alt="Figure 6: no antibiotics bacterial number variation through time with PLL"><figcaption></figcaption>Figure 6: no antibiotics bacterial number variation through time with PLL</figure>
                          <p>After mathematical simulation we found that the adhesion rate of PAO1 by polylysine after
-                             treatment of PLL is increased, while for E. coli, the adhesion quantity rise, not fall.</p>
+                             treatment of PLL is increased, while for E. coli, the adhesion quantity rise, not
+                            fall.</p>
                          <p><img src="https://static.igem.org/mediawiki/2015/0/0d/USTC-PAO1.png" alt="图片名称"></p>
-                         <p><em>As for HCB1</em>, a kind of <em>E. coli</em> and the number of bacteria inside observed
+                         <p><em>As for HCB1</em>, a kind of <em>E. coli</em> and the number of bacteria inside
-                             field is stable and relatively declined because of its lack of capability of
+                             observed field is stable and relatively declined because of its lack of capability of
-                             adhesion.<br><img src="https://static.igem.org/mediawiki/2015/6/6f/Development22.png" alt="图片名称">
+                             adhesion.<br><img src="https://static.igem.org/mediawiki/2015/6/6f/Development22.png"
-                        </p>
+                                              alt="图片名称"></p>
-                         <p>After treated with 20ug/mL polylysine, adhesive bacteria number obviously increased:<br><img
+                         <p>After treated with 20ug/mL polylysine, adhesive bacteria number obviously
-                                src="https://static.igem.org/mediawiki/2015/9/96/4141414.png" alt="图片名称"></p>
+                            increased:<br><img src="https://static.igem.org/mediawiki/2015/9/96/4141414.png" alt="图片名称">
+                        </p>
                          <p>Modeling on adhesion process strictly demonstrated the adhesion assay fits Langmuir
                              absorption isoform.</p>
-                         <p><img src="https://static.igem.org/mediawiki/2015/a/a9/Ustc-bacterial_HCB1.png" alt="图片名称"><br><img
+                         <p><img src="https://static.igem.org/mediawiki/2015/a/a9/Ustc-bacterial_HCB1.png"
-                                src="https://static.igem.org/mediawiki/2015/d/d8/20150901022.png" alt="图片名称"><br>Simulation
+                                alt="图片名称"><br><br>Simulation result:<br>\(\sigma =K\sigma _{0}\times (1-e^{-\frac{K_{\alpha}CV_{z}}{K\sigma _{0}}(t-t_{0})})\)<br>Constants
-                            result:<br><img src="https://static.igem.org/mediawiki/2015/8/86/20150903028.png" alt=""><br>Constants
                              value and details:<br><img src="https://static.igem.org/mediawiki/2015/6/66/20150903029.png"
                                                         alt=""></p>
-                         <p>To know more about our modeling, please check <a href="http://tower.im">Modeling: Adhesion
+                         <p>To know more about our modeling, please check <a href="https://2015.igem.org/Team:USTC/Modeling#Adhesion-Dynamics">Modeling:
-                             Assay Modeling</a></p>
+                             Adhesion Dynamics</a></p>
-                         <p>This significant reverse of bacterial adhesion tendency stronly proved that polylysine has
+                         <p>This significant reverse of bacterial adhesion tendency stronly proved that polylysine
-                             effective adhesive ability for SPRING.</p>
+                             has effective adhesive ability for SPRING.</p>
-                         <p>After we confirmed the feasibility of PLL treatment for bacterial adhesion, then the treating
+                         <p>After we confirmed the feasibility of PLL treatment for bacterial adhesion, then the
-                             time should be taken into consideration because only in the case of stable adhesion is
+                             treating time should be taken into consideration because only in the case of stable
-                             effective for SPRING to gather effective and stable data.</p>
+                             adhesion is effective for SPRING to gather effective and stable data.</p>
-                         <p>As the plot illustrates, bacteria number is growing at the beginning of PLL treatment because
+                         <p>As the plot illustrates, bacteria number is growing at the beginning of PLL treatment
-                             of opening strong electrostatic adsorption derived from PLL. And after 1min, the number of
+                             because of opening strong electrostatic adsorption derived from PLL. And after 1min, the
-                             bacteria become stable, and there is nearly no difference on bacteria number comparing after
+                             number of bacteria become stable, and there is nearly no difference on bacteria number
-min treatment to after 5 min treatment.</p>
+                             comparing after 1 min treatment to after 5 min treatment.</p>
                          <p>Consequently, we recommended that PLL treatment after 5 min would be a promising set for
                              SPRING to output stable data.</p>
-                         <p><strong>Best Measurement Interval</strong></p>
+                         <h4 id="Best_Measurement_Interval" class="scrollspy">Best Measurement Interval</h4>
-                         <p>According to chemotaxis, bacteria responsing to surrounding pressure or beneficits will be
+                         <p>According to chemotaxis, bacteria responsing to surrounding pressure or beneficits will
-                             presented as change of bacteria movement. Here we analyzed bacterial movement data treating
+                             be presented as change of bacteria movement. Here we analyzed bacterial movement data
-                             with different antibiotics concentration, specificly, chloromycetin concentration. And we
+                             treating with different antibiotics concentration, specificly, chloromycetin
-                            gathered bacterial movement data just after antibiotics treatment(0s), after 1 min treatment
+                            concentration. And we gathered bacterial movement data just after antibiotics
-                            and after 5 min treatment, and we again recorded all pictures in the following 18 s to get
+                            treatment(0s), after 1 min treatment and after 5 min treatment, and we again recorded
-                            the dynamic data. Then, we concluded from the percentage of bacterial movement as an
+                            all pictures in the following 18 s to get the dynamic data. Then, we concluded from the
-                            important data to intercept the effect of bacterial response on antibiotics.</p>
+                            percentage of bacterial movement as an important data to intercept the effect of
+                            bacterial response on antibiotics.</p>
                          <p>The measurement assay is illustrated as following:</p>
                          <p><em>PLL(-)</em></p>
-                         <table>
+                         <table class="striped">
                              <thead>
                              <tr>
@@ Line 342: / Line 737: @@
                          </table>
                          <p><em>PLL(+,20)</em></p>
-                         <table>
+                         <table  class="striped">
                              <thead>
                              <tr>
@@ Line 413: / Line 808: @@
                          </table>
                          <p>When bacteria are treated with PLL, the movement of bacteria is been limited for strong
-                             adhesion, after 5 min treatment, the proportion of movement decreased through time.<br><img
+                             adhesion, after 5 min treatment, the proportion of movement decreased through
-                                    src="https://static.igem.org/mediawiki/2015/9/9b/-ustc-nosd.png" alt="图片名称"></p>
+                            time.<br><img src="https://static.igem.org/mediawiki/2015/9/9b/-ustc-nosd.png" alt="图片名称"></p>
-                         <p>However, when bacteria are not treated with PLL, incubated in 1ug/mL chloromycetin solution,
+                         <p>However, when bacteria are not treated with PLL, incubated in 1ug/mL chloromycetin
-                             we got the movement percentage of bacteria as following:</p>
+                             solution, we got the movement percentage of bacteria as following:</p>
                          <p><img src="https://static.igem.org/mediawiki/2015/c/ca/Results-0.png" alt="图片名称"></p>
@@ Line 428: / Line 823: @@
                          <p>This is the bacterial movement percentage variation with time, treated with 0.1 ug/mL
-                             chloromycetin solution. Antibiotics pressure is not that strong, consequently, the movement
+                             chloromycetin solution. Antibiotics pressure is not that strong, consequently, the
-                             of bacteria is not ignited significantly.</p>
+                             movement of bacteria is not ignited significantly.</p>
                          <p><img src="https://static.igem.org/mediawiki/2015/b/b0/Results-1.png" alt="图片名称"></p>
-                         <p>When it comes to 0.5 ug/mL chloromycetin solution, the proportion of the moving bacteria is
+                         <p>When it comes to 0.5 ug/mL chloromycetin solution, the proportion of the moving bacteria
-                             increased. And as a matter of fact, the increase is quite corresponding through time,
+                             is increased. And as a matter of fact, the increase is quite corresponding through time,
                              reflecting bacteria impressive response on antibiotics substance.<br><img
-                                     src="https://static.igem.org/mediawiki/2015/3/32/Ustc-2015-results-2.png" alt="图片名称"></p>
+                                     src="https://static.igem.org/mediawiki/2015/3/32/Ustc-2015-results-2.png" alt="图片名称">
+                        </p>
                          <p>What if the concentration of chloromycetin solution up to 1 ug/mL?<br><img
                                  src="https://static.igem.org/mediawiki/2015/0/0a/Ustc-2015-results-4.png" alt="图片名称"><br>A
-                             quite linear increase of the proportion of the moving bacteria with time is observed.</p>
+                             quite linear increase of the proportion of the moving bacteria with time is observed.
+                        </p>
-                         <p>Consequently, according to our experiment, genetically naturally bacteria could respond to
+                         <p>Consequently, according to our experiment, genetically naturally bacteria could respond
-                             chloromycetin solution from 0.1 ug/mL to more than 1ug/mL, which is quite promising after
+                             to chloromycetin solution from 0.1 ug/mL to more than 1ug/mL, which is quite promising
-                             genetic modification.</p>
+                             after genetic modification.</p>
                          <p><img src="https://static.igem.org/mediawiki/2015/c/c3/Finalresult.png" alt="图片名称"></p>
+                    </div>
+                </div>
+                <div class="col hide-on-small-only m3">
+                    <div class="toc-wrapper pinned">
+                        <ul class="section table-of-contents">
+                            <li>
+                                <a href="#characterization-of-optimal-conditions-on-polylysine-pll-coated-assay">Characterization
+                                    of Optimal Conditions on Polylysine(PLL) Coated Assay</a>
+                            </li>
+                            <div class="divider"></div>
+                            <li>
+                                <a href="#Best_Bacterial_Developmental_Interval_Along_with_Recommended_Dilution_Conditions">Best
+                                    Bacterial Developmental Interval Along with Recommended Dilution Conditions</a>
+                            </li>
+                            <div class="divider"></div>
+                            <li>
+                                <a href="#Best_PLL-Coated_Concentration_and_Pre-treatment_Time">Best PLL-Coated
+                                    Concentration and Pre-treatment Time.</a>
+                            </li>
+                            <div class="divider"></div>
+                            <li>
+                                <a href="#Adhesion_Assay_with_PLL_treatment">Adhesion Assay with PLL treatment</a>
+                            </li>
+                            <div class="divider"></div>
+                            <li>
+                                <a href="#Best_PLL-Coated_Time">Best PLL-Coated Time</a>
+                            </li>
+                            <div class="divider"></div>
+                            <li>
+                                <a href="#Best_Measurement_Interval">Best Measurement Interval</a>
+                            </li>
+                        </ul>
+                    </div>
+                </div>
+            </div>
+        </div>
+        <div id="Film_Determination" class="row">
+            <div class="card hoverable">
+                <div class="col s12 m9">
+                    <div class="card-content">
+                        <p>Let's do some crasy science this time! We'll show you how we test and select the most
+                            important part in our project, the special artificial original film!</p>
+                        <h4 id="modeling-guide-us-choose-the-candidates" class="scrollspy">Modeling guide us choose the
+                            candidates</h4>
+                        <p>In our modeling, we start with single bacteria force, analyse the interaction between
+                            bacteria and film, and propose the request of Young modulus (&lt;1GPa) of film
+                            eventually.</p>
+                        <p>And there are various film candidates come into the front.</p>
+                        <h4 id="processing-film" class="scrollspy">Processing Film</h4>
+                        <p>The film must be kind of soft and one of the surface should have enough reflectance while
+                            another surface should have the ability to adhere to bacteria.<br>In fact, we don't hear any
+                            of this type of film. So we need to produce the film by our own.</p>
+                        <h4 id="film-i" class="scrollspy">Film I</h4>
+                        <p><strong>Low Pressure Polyethylene</strong></p>
+                        <figure><img
+                                src="https://static.igem.org/mediawiki/2015/e/ee/Ustc-filmcandid1.png"
+                                alt="Figure 1: Film I, polyethylene"><figcaption>Figure 1: Film I, polyethylene</figcaption></figure>
+                        <p>Low pressure polyethylene is soft enough, and is too soft.</p>
+                        <p>We use aerosol paint cover one of the surface. That can make the other surface of the film
+                            will become a reflect surface. And we use 400ul 20ug/ml PLL coating the same surface at the
+                            temperature of 4℃ to make this surface has the ability to adhere to bacteria.</p>
+                        <p>But we fail with <strong>film I</strong> in the end that we can't get interference fringes.
+                            Because the surface of low pressure polyethylene is not as smooth as we want, that will
+                            cause a diffuse reflection. Thus we can not get interference inescapably.</p>
+                        <p>So we need the film becoem more smooth and more elastic, and then come out <strong>film
+                            II-Rubbers</strong></p>
+                        <h4 id="film-ii" class="scrollspy">Film II</h4>
+                        <p><strong>Rubbers-Condom</strong></p>
+                        <figure><img
+                                src="https://static.igem.org/mediawiki/2015/b/b7/Ustc-filmcandid3.jpeg"
+                                alt="Figure 2: Film II, condom"><figcaption>Figure 2: Film II, condom</figcaption></figure>
+                        <p>If you want the material smooth enough and thick enough and elastic enough, that is, of
+                            cause, <strong>condom!</strong></p>
+                        <p>When we plan to use paint cover, the truth give us a hard hit. Because the film shrink too
+                            much when we spray paint.</p>
+                        <p>And we try silver mirror reaction as replacement. But still fail Because the ammonia erosion
+                            is too severe.</p>
+                        <p>Finally we find the final film candidate that we missed-<strong>Glass</strong></p>
+                        <h4 id="film-iii" class="scrollspy">Film III</h4>
+                        <p><strong>Cover slip</strong></p>
+                        <figure><img
+                                src="https://static.igem.org/mediawiki/2015/e/ef/Ustc-filmcandid2.png"
+                                alt="Figure 3: Film III, Cover slip"><figcaption>Figure 3: Film III, Cover slip</figcaption></figure>
+                        <p>The Young modulus of Glass is about 50GPa. But according to our <strong>adhesion
+                            assay</strong> result and modeling, there will several numbers of fringes changes in the
+                            experiment.</p>
+                        <p>So we spraying it, coating it, testing it, and we made it!<br>The glass have a great
+                            potential to reflect, so we can get interference very easy.<br>With modeling guidance, we
+                            can know the relation between fringes changes and antibiotics concentration.<br>\(A_{0}+\frac{B_{0}}{A}=\frac{1}{\Delta N}\)</p>
+                        <p>Then we want to develop a calibration with three point.</p>
+                        <figure><img
+                                src="https://static.igem.org/mediawiki/2015/e/e4/20150918066.jpg"
+                                alt="Figure 5: Experimental results of calibration"><figcaption>Figure 5: Experimental results of calibration</figcaption></figure>
+                        <p>The fitting result indicate that
+                            our modeling and normalization operation was exactly correct and effective. <strong>OUR
+                                CRAZY MIND HIT OUR MODEL, AND OUR MODEL EXACYLY HIT OUR RESULT! WE LOVE
+                                SCIENCE!</strong></p>
+                        <p>That means the <strong>Processed Glass</strong> worth the name of <strong>special artificial
+                            original film!</strong></p>
+                    </div>
+                </div>
+                <div class="col hide-on-small-only m3">
+                    <div class="toc-wrapper pinned">
+                        <ul class="section table-of-contents">
+                            <li>
+                                <a href="#modeling-guide-us-choose-the-candidates">Modeling guide us choose the candidates</a>
+                            </li>
+                            <div class="divider"></div>
+                            <li>
+                                <a href="#processing-film">Processing Film</a>
+                            </li>
+                            <div class="divider"></div>
+                            <li>
+                                <a href="#film-i">Film I</a>
+                            </li>
+                            <div class="divider"></div>
+                            <li>
+                                <a href="#film-ii">Film II</a>
+                            </li>
+                            <div class="divider"></div>
+                            <li>
+                                <a href="#film-iii">Film III</a>
+                            </li>
+                        </ul>
+                    </div>
+                </div>
+            </div>
+        </div>
+        <div id="Calibration" class="row">
+            <div class="card hoverable">
+                <div class="col s12 m9">
+                    <div class="card-content">
+                        <p>You can detect various of kinds of material in solution, as long as you follow our standard to make the engineered bacteria. See our future NDM improvement plan at <a href="https://2015.igem.org/Team:USTC/Achievements#Future-NDM">Future NDM</a></p>
+                        <p>There is one thing to be sure is that whatever bacteria you used, you need to build calibration ruler to measure the concentration quantificationally. According to our modeling in calibration we need to confirm three constants <strong>'A0', 'B0', 'n'</strong>.</p>
+                        <p>\(A_{0}+\frac{B_{0}}{A}=\frac{1}{\Delta N}\)</p>
+                        <p>That means we need to test at least three point to confirm the formula.</p>
+                        <h4 id="establishement-of-the-calibration-ruler" class="scrollspy">Establishement of the Calibration Ruler</h4>
+                        <p>To build a calibration ruler between <strong>chloramphenicol</strong> concentration and</p>
+                        <p>interference fringes pattern.</p>
+                        <p>In this case we use our <em>E. coli</em> BL21 with T7-OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>), a type of bacteria in the best perfomance during genetically modification.(See our modification at <a href="https://2015.igem.org/Team:USTC/Results#Permeability-Improvement">Results-Permeability Improvement</a>). According to our experiment results, we found the <em>E. coli</em> BL21 with T7-OprF (<a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a>) was more sensitive to chloramphenicol.</p>
+                        <p>And according to <strong>adhesion pre-experiment</strong> we chose the concentration range with 0.5ug/ml~5ug/ml in the experiment.</p>
+                        <p>The changes of fringe number and test concentration shown below(take three pictures in a row with the time interval 20s),</p>
+                        <table>
+                            <thead>
+                                <tr>
+                                    <th>Time(s)</th>
+                                    <th>5.0ug/ml(Fringe number)</th>
+                                    <th>1.0ug/ml(Fringe number)</th>
+                                    <th>0.5ug/ml(Fringe number)</th>
+                                </tr>
+                            </thead>
+                            <tbody>
+                                <tr>
+                                    <td>0</td>
+                                    <td>42/43</td>
+                                    <td>51/49/52</td>
+                                    <td>44/43</td>
+                                </tr>
+                                <tr>
+                                    <td>20</td>
+                                    <td>-</td>
+                                    <td>50/51/50</td>
+                                    <td>-</td>
+                                </tr>
+                                <tr>
+                                    <td>40</td>
+                                    <td>-</td>
+                                    <td>50/46</td>
+                                    <td>-</td>
+                                </tr>
+                                <tr>
+                                    <td>60</td>
+                                    <td>-</td>
+                                    <td>48/48/50</td>
+                                    <td>45</td>
+                                </tr>
+                                <tr>
+                                    <td>80</td>
+                                    <td>-</td>
+                                    <td>48/49/49</td>
+                                    <td>-</td>
+                                </tr>
+                                <tr>
+                                    <td>100</td>
+                                    <td>-</td>
+                                    <td>51/53/49</td>
+                                    <td>45</td>
+                                </tr>
+                                <tr>
+                                    <td>120</td>
+                                    <td>42/43</td>
+                                    <td>-</td>
+                                    <td>-</td>
+                                </tr>
+                                <tr>
+                                    <td>140</td>
+                                    <td>-</td>
+                                    <td>51/48/52</td>
+                                    <td>-</td>
+                                </tr>
+                                <tr>
+                                    <td>160</td>
+                                    <td>-</td>
+                                    <td>-</td>
+                                    <td>41</td>
+                                </tr>
+                                <tr>
+                                    <td>180</td>
+                                    <td>-</td>
+                                    <td>48/52/54</td>
+                                    <td>-</td>
+                                </tr>
+                                <tr>
+                                    <td>200</td>
+                                    <td>48</td>
+                                    <td>55/47/49</td>
+                                    <td>43/43</td>
+                                </tr>
+                                <tr>
+                                    <td>220</td>
+                                    <td>43/47</td>
+                                    <td>51/52</td>
+                                    <td>45</td>
+                                </tr>
+                                <tr>
+                                    <td>240</td>
+                                    <td>48/46</td>
+                                    <td>53/51</td>
+                                    <td>44</td>
+                                </tr>
+                                <tr>
+                                    <td>starting average</td>
+                                    <td>42.5</td>
+                                    <td>50.5</td>
+                                    <td>43.5</td>
+                                </tr>
+                                <tr>
+                                    <td>ending average</td>
+                                    <td>46.4</td>
+                                    <td>51.75</td>
+                                    <td>44.2</td>
+                                </tr>
+                                <tr>
+                                    <td>ΔN</td>
+                                    <td>3.9</td>
+                                    <td>1.25</td>
+                                    <td>0.7</td>
+                                </tr>
+                            </tbody>
+                        </table>
+                        <p>Then we can fit this data with calibration formula, assume <strong>n=3</strong>(through trying different value of n get the best value of n) then get the simplified formula:</p>
+                        <p>\(A_{0}+\frac{B_{0}}{A}=\frac{1}{\Delta N}\)</p>
+                        <p>Then the fitting result illstruates as,</p>
+                        <p><img src="https://static.igem.org/mediawiki/2015/e/e4/20150918066.jpg" alt=""></p>
+                        <p>And we can get the value of unknown constants:</p>
+                        <table>
+                            <thead>
+                                <tr>
+                                    <th>constant</th>
+                                    <th>value</th>
+                                </tr>
+                            </thead>
+                            <tbody>
+                                <tr>
+                                    <td>A0</td>
+                                    <td>0.1339</td>
+                                </tr>
+                                <tr>
+                                    <td>B0</td>
+                                    <td>0.6513</td>
+                                </tr>
+                            </tbody>
+                        </table>
+                        <p><strong>What an amazing result! </strong> The fitting result indicated that our modeling and normalization operation was exactly correct and effective. <strong>OUR CRAZY MIND HIT OUR MODEL, AND OUR MODEL EXACYLY HIT OUR RESULT!</strong> <strong>WE LOVE SCIENCE!</strong></p>
+                        <div class="divider"></div>
+                        <h4 id="reverification-of-calibration" class="divider">Reverification of Calibration</h4>
+                        <p>There may some arguement on our accuracy on calibration. So we need to conduct another experiment to reverficate our calibration ruler.</p>
+                        <p>In order to test whether the calibration is correct and to confirm the coefficients, we choose two concentration as check point.</p>
+                        <p>And use SPRING to measure its concentration. The real concentration and experiment results shown in table:</p>
+                        <table>
+                            <thead>
+                                <tr>
+                                    <th>time(s)</th>
+                                    <th>1.8ug/ml(Fringe number)</th>
+                                    <th>3.2ug/ml(Fringe number)</th>
+                                </tr>
+                            </thead>
+                            <tbody>
+                                <tr>
+                                    <td>0</td>
+                                    <td>78/80</td>
+                                    <td>77/76/77</td>
+                                </tr>
+                                <tr>
+                                    <td>20</td>
+                                    <td>77/77</td>
+                                    <td>79/77</td>
+                                </tr>
+                                <tr>
+                                    <td>40</td>
+                                    <td>78</td>
+                                    <td>75/72</td>
+                                </tr>
+                                <tr>
+                                    <td>60</td>
+                                    <td>79</td>
+                                    <td>74</td>
+                                </tr>
+                                <tr>
+                                    <td>80</td>
+                                    <td>81</td>
+                                    <td>75/75</td>
+                                </tr>
+                                <tr>
+                                    <td>100</td>
+                                    <td>75</td>
+                                    <td>72/73</td>
+                                </tr>
+                                <tr>
+                                    <td>120</td>
+                                    <td>80</td>
+                                    <td>74/74</td>
+                                </tr>
+                                <tr>
+                                    <td>140</td>
+                                    <td>83/82</td>
+                                    <td>71/75</td>
+                                </tr>
+                                <tr>
+                                    <td>160</td>
+                                    <td>81/79</td>
+                                    <td>72/76</td>
+                                </tr>
+                                <tr>
+                                    <td>180</td>
+                                    <td>82/82</td>
+                                    <td>75/74</td>
+                                </tr>
+                                <tr>
+                                    <td>200</td>
+                                    <td>82/81</td>
+                                    <td>75</td>
+                                </tr>
+                                <tr>
+                                    <td>220</td>
+                                    <td>80/82</td>
+                                    <td>74/74/75</td>
+                                </tr>
+                                <tr>
+                                    <td>240</td>
+                                    <td>81/80</td>
+                                    <td>74/75/74</td>
+                                </tr>
+                                <tr>
+                                    <td>starting average</td>
+                                    <td>79</td>
+                                    <td>77.2</td>
+                                </tr>
+                                <tr>
+                                    <td>ending average</td>
+                                    <td>81</td>
+                                    <td>74.3</td>
+                                </tr>
+                                <tr>
+                                    <td>ΔN</td>
+                                    <td>2.0</td>
+                                    <td>2.9</td>
+                                </tr>
+                                <tr>
+                                    <td>experiment concentration</td>
+                                    <td>1.80ug/ml</td>
+                                    <td>3.09ug/ml</td>
+                                </tr>
+                            </tbody>
+                        </table>
+                        <p><img src="https://static.igem.org/mediawiki/2015/1/13/USTC_Result_pic.png" alt=""></p>
+                        <p>The result showed that the experiment concentration is <strong>EXTREMELY CLOSE</strong> to the real concentration, which means <strong>OUR NDM IS ABLE TO TELL ANTIBIOTIC CONCENTRATION IN VERY ACCURATE WAY.</strong></p>
+                        <p>By the way, <strong>DO NOT FORGET,  </strong> operating our NDM only cost you <strong>100 SECONDS</strong>.</p>
+                        <p>Yes, such fast and accurate hardware detecting antibiotics, <strong>that is our NDM</strong>, <strong>ONLY OUR NDM</strong>.</p>
+                        <p><em>Note:</em> To get the program of our calibration analysis, please visit <a href="https://2015.igem.org/Team:USTC/Software">Software</a>. To know more information on how we analyzed our results, please visit <a href="https://2015.igem.org/Team:USTC/Modeling#Calibration">Modeling: Calibration</a>. To see our final measurement report with theoretical analysis, please visit <a href="https://2015.igem.org/Team:USTC/Measurement">Measurement</a>.</p>
+                    </div>
+                </div>
+                <div class="col hide-on-small-only m3">
+                    <div class="toc-wrapper pinned">
+                        <ul class="section table-of-contents">
+                            <li>
+                                <a href="#establishement-of-the-calibration-ruler">Establishement of the Calibration Ruler</a>
+                            </li>
+                            <li>
+                                <a href="#reverification-of-calibration">Reverification of Calibration</a>
+                            </li>
+                        </ul>
                      </div>
                  </div>
@@ Line 454: / Line 1,271: @@
      </div>
+</div>
 </div>
 </html>
 {{USTC/footer}}

Antibiotics Concentration(ug/mL)	Incubation Time(s)
0	0
0	60
0	120
0	180
0	240
0	300
0.1	0
0.1	60
0.1	300
0.5	0
0.5	60
0.5	300
1	0
1	60
1	300

Antibiotics Concentration(ug/mL)	Incubation Time(s)
0	0
0	60
0	120
0	180
0	240
0	300
0.1	0
0.1	60
0.1	300
0.5	0
0.5	60
0.5	300
1	0
1	60
1	300

Antibiotics Concentration(ug/mL)	Incubation Time(s)
0	0
0	60
0	120
0	180
0	240
0	300
0.1	0
0.1	60
0.1	300
0.5	0
0.5	60
0.5	300
1	0
1	60
1	300