Difference between revisions of "Team:USTC/Results"

Revision as of 01:11, 19 September 2015

Overview
Permeability Improvement
Chemotaxis & ROSE
Adhesion Assay
Film Determination

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!

Want to get them now? Click their title directly!

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.

Figure 1: Extraction of SCVE fragement

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

Figure 2: Extraction of OprF fragement

T7 is a strong promoter which we got form Parts Registry. T7 will be used to trigger the expression of both OprF and SCVE.
Cas9 is got from 2015 Part Distribution.

Figure 4: Extraction of Cas9 from parts

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

Figure 9: ONPG reaction

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.

Figure 10: ONPG assay schematic show

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.

Figure 11: ONPG assay, absorption OD406

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.

Figure 14: NPN uptake result-1

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.

Figure 15: NPN uptake result in average

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 Assa
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.

Figure 2: fragements extraction of cheZ

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.

Figure 5: Growth characteristics of BL21 WT with lac-cheZ [BBa_K1593997](http://parts.igem.org/Part:BBa_K1593997) 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.
Figure 6: characterization of cheZ expression by SDS-PAGE
See protocol in Protocols: SDS-PAGE

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 Adhesion: Polylysine.

To get the final PLL-coated protocol, check Protocols: PLL Coated Assay 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: Bacterial number and movement analysis.

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.

图片名称

Simulation result:

Constants value and details:

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

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.
图片名称

However, when bacteria are not treated with PLL, incubated in 1ug/mL chloromycetin solution, we got the movement percentage of bacteria as following:

图片名称

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?
图片名称
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
Figure 1: Film I, 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
Figure 2: Film II, 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
Figure 3: 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.
Figure 4 :Relation between fringes changes and antibiotics concentration

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

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+                        <p>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:</p>
+                        <ul>
+                            <li><p>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.</p>
+                            </li>
+                            <li><p>Chemotaxis Modification: This section introduces results of chemotaxis
+                                engineering.</p>
+                            </li>
+                            <li><p>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.</p>
+                            </li>
+                            <li><p>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!</p>
+                            </li>
+                            <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>
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+                    <div class="col s12 m9">
+                        <div class="card-content">
+                            <h4 id="Extraction_of_Permeability_Improvement_Fragements" class="scrollspy">Extraction of
+                                Permeability Improvement Fragements</h4>
+                            <p>We successfully get
+                                gene fragements that are used for improving bacterial permeability, which including,
+                            </p>
+                            <ul>
+                                <li>SCVE, a viroporin originally from SARS virus, is synthesized by Sangon Co. ctd.</li>
+                            </ul>
+                            <p><img src="https://static.igem.org/mediawiki/2015/1/1d/Ustc-scve.jpeg"
+                                    alt="Figure 1: Extraction of SCVE fragement"></p>
+                            <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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/6/6d/Ustc-t7oprf_lac-chez.jpeg"
+                                    alt="Figure 2: Extraction of OprF fragement"></p>
+                            <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><img
+                                            src="https://static.igem.org/mediawiki/2015/7/7a/Ustc-T7.jpeg"
+                                            alt="Figure 3: T7 extraction from parts"></p>
+                                </li>
+                                <li><p>Cas9 is got from 2015 Part Distribution.</p>
+                                </li>
+                            </ul>
+                            <p><img src="https://static.igem.org/mediawiki/2015/1/1c/USTC-Cas9.png"
+                                    alt="Figure 4: Extraction of Cas9 from parts"></p>
+                            <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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/5/54/USTC-gRNA_extraction.png"
+                                    alt="Figure 5: Extraction of gRNA-AceB and gRNA-EmrE, two fragments are synthesized by Sangon Biotech Company">
+                            </p>
+                            <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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/e/ed/Ustc-scve_jp_t7.jpeg"
+                                    alt="Figure 6: Construction of T7-SCVE ([BBa_K1593667](http://parts.igem.org/Part:BBa_K1593667))">
+                            </p>
+                            <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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/6/6d/Ustc-t7oprf_lac-chez.jpeg"
+                                    alt="Figure 7: Construction of T7-OprF ([BBa_K1593210](http://parts.igem.org/Part:BBa_K1593210))">
+                            </p>
+                            <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://tower.im">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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/d/d8/Ustc-growth2.png"
+                                    alt="Figure 8: Bacteria growth characterization"></p>
+                            <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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/5/54/ONPG-ustc-sche.png"
+                                    alt="Figure 9: ONPG reaction"></p>
+                            <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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/3/32/Ustc-board.png"
+                                    alt="Figure 10: ONPG assay schematic show"></p>
+                            <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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/5/52/Ustc-onpg88.png"
+                                    alt="Figure 11: ONPG assay, absorption OD406"></p>
+                            <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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/0/00/Ustc-onpg55.png"
+                                    alt="Figure 12: ONPG assay, absorption OD406 in average"></p>
+                            <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>
+                            <p><img
+                                    src="https://static.igem.org/mediawiki/2015/1/16/Ustc-npn.gif"
+                                    alt="Figure 13: Schematic Structure of N-Phenyl-1-naphthylamine, NPN"></p>
+                            <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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/e/e5/Ustc-npn1.png"
+                                    alt="Figure 14: NPN uptake result-1"></p>
+                            <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>
+                            <p><img src="https://static.igem.org/mediawiki/2015/1/1a/Ustc-npn2.png"
+                                    alt="Figure 15: NPN uptake result in average "></p>
+                            <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>
+                        </div>
+                    </div>
+                    <div class="col hide-on-small-only m3">
+                        <div class="toc-wrapper pinned">
+                            <ul class="section table-of-contents">
+                                <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 Assa</a>
-            <div class="row center" style="margin-top:20px;">
+                                    <div class="divider"></div>
-            <a href="https://static.igem.org/mediawiki/2015/b/bd/USTCUSTC_Manual.pdf" id="download-button" class="btn-large waves-effect waves-light blue">Get it now!</a>
+                                <li>
-          </div>
+                                    <a href="#bibliography">Bibliography</a>
+                                </li>
-          </div>
+                            </ul>
+                        </div>
+                    </div>
+                </div>
+            </div>
          </div>
-      </div>
+        <div id="Chemotaxis_Modification_and_ROSE" class="row">
-    </div>
+            <div class="card hoverable">
+                <div class="row">
+                    <div class="col s12 m9">
+                        <div class="card-content">
+                            <h4 id="Genetic_Fragments_Extraction" class="scrollspy">Genetic Fragments Extraction</h4>
+                            <ul>
+                                <li>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.
+                                </li>
+                            </ul>
+                            <p><img src="https://static.igem.org/mediawiki/2015/7/7e/Ustc-micf_soxs_xh.jpeg"
+                                    alt="Figure 1: fragements extraction of micF and SoxS"></p>
+                            <ul>
+                                <li>cheZ, this is the chemotaxis modified fragment.</li>
+                            </ul>
+                            <p><img src="https://static.igem.org/mediawiki/2015/8/8c/Ustc-chez.jpeg"
+                                    alt="Figure 2: fragements extraction of cheZ"></p>
+                            <ul>
+                                <li>BBa_R0010, this is promoter of lac, which triggers the expression of cheZ.</li>
+                            </ul>
+                            <p><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)">
+                            </p>
-    <div id="Make-Your-Own" class="row">
+                            <p><strong>Construction of plasimid with lac-cheZ <a
-      <div class="card hoverable">
+                                    href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a></strong></p>
-        <div class="col s12 m9">
-          <div class="card-content">
-          <h4 id="Materials" class="scrollspy">Materials</h4>
-          <p>To accomplish SPRING, you need prepare those material showed below:</p>
-          <table>
-            <thead>
-              <tr>
-                <th>Materials</th>
-                <th>Number</th>
-                <th>Price in estimation($)</th>
-              </tr>
-            </thead>
-            <tbody>
-              <tr>
-                <td>12 in 1 display</td>
-                <td>1</td>
-                <td>92.08</td>
-              </tr>
-              <tr>
-                <td>CSI Interface Camera</td>
-                <td>1</td>
-                <td>13.88</td>
-              </tr>
-              <tr>
-                <td>Camera Bracket</td>
-                <td>1</td>
-                <td>1.42</td>
-              </tr>
-              <tr>
-                <td>3V laser head</td>
-                <td>2</td>
-                <td>0.31</td>
-              </tr>
-              <tr>
-                <td>5V laser head module</td>
-                <td>2</td>
-                <td>1.73</td>
-              </tr>
-              <tr>
-                <td>Arduino UNO R3</td>
-                <td>1</td>
-                <td>4.67</td>
-              </tr>
-              <tr>
-                <td>Universal circuit board</td>
-                <td>5</td>
-                <td>0.41</td>
-              </tr>
-              <tr>
-                <td>Breadboard</td>
-                <td>2</td>
-                <td>1.75</td>
-              </tr>
-              <tr>
-                <td>Dupont line</td>
-                <td>15</td>
-                <td>4.25</td>
-              </tr>
-              <tr>
-                <td>12V lithium battery</td>
-                <td>1</td>
-                <td>15.72</td>
-              </tr>
-              <tr>
-                <td>12.6V 3A Chargers</td>
-                <td>1</td>
-                <td>6.06</td>
-              </tr>
-              <tr>
-                <td>DC-DC step-down module</td>
-                <td>2</td>
-                <td>5.04</td>
-              </tr>
-              <tr>
-                <td>PP board</td>
-                <td>1</td>
-                <td>7.71</td>
-              </tr>
-              <tr>
-                <td>Miniature hand drill</td>
-                <td>1</td>
-                <td>20.46</td>
-              </tr>
-            </tbody>
-          </table>
-          <div class="divider"></div>
-          <h4 id="Protection-Guide" class="scrollspy">Protection Guide</h4>
-          <p><img src="https://static.igem.org/mediawiki/2015/c/ca/Ustc-myo15.png"></p>
-          <p>When you are making NDM, many tools are needed, as well as touching bacteria solution. Consequently, we provided four recommended protective measures for everyone. Please pay attention to personal safety when doing experiment:</p>
-          <p><img src="https://static.igem.org/mediawiki/2015/e/eb/Ustc-myo11.png"></p>
-          <ul>
-            <li>Level A: Fundamental protective measure when producing NDM and conducting experiment. You may wear lab coat. When conducting experiments and assemble NDM, level A should be satisfied always.</li>
-          </ul>
-          <p><img src="https://static.igem.org/mediawiki/2015/1/11/Ustc-myo12.png"></p>
-          <ul>
-            <li>Level B: In level B, personnel should not only wear lab coat, but wear nitrile gloves or latex gloves, which are necessary when touching bacteria solution. This procedure is the first step for detection.</li>
-          </ul>
-          <p><img src="https://static.igem.org/mediawiki/2015/5/54/Ustc-myo13.png"></p>
-          <ul>
-            <li>Level C: Level C is the standard protocol for personnels when drilling. In addtion to level A, you should wear goggles. Goggles would protect your eyes when you are drilling plexiglass. And please do remember ROLL UP YOUR SLEEVES to prevent possible hurts and DO NOT WEAR GLOVES because it is not convenient when drilling.</li>
-          </ul>
-          <p><img src="https://static.igem.org/mediawiki/2015/9/91/Ustc-myo14.png"></p>
-          <ul>
-            <li>Level D: This is the most complicated protective measure for spraing painting procedure. One should wear lab coats, along with goggles, masks and gloves to toally protect oneself considering extreme pungent odor volatilizing when painting.</li>
-          </ul>
-          <div class="divider"></div>
-          <h4 id="Tools" class="scrollspy">Tools</h4>
-          <p><img src="https://static.igem.org/mediawiki/2015/0/01/Ustc-myo1.png"></p>
-          <ul>
-            <li>Drill is used for cutting acrylic materials for assemble bracket.</li>
-            <li>Electric soldering iron, for welding electric circuit.</li>
-            <li>Glue gun is needed for adhesion between each other acrylic materials.</li>
-          </ul>
-          <div class="divider"></div>
-          <h4 id="Display-module" class="scrollspy">Display module</h4>
-          <p><img src="https://static.igem.org/mediawiki/2015/5/52/Ustc-myo2.png"></p>
-          <p>The display module contains 12 in 1 display with processor and buttons. Users are able to control the power, contrast and other display effects through buttons beside the display.</p>
-          <div class="divider"></div>
-          <h4 id="Raspberry-Pi" class="scrollspy">Raspberry Pi</h4>
-          <p><img src="https://static.igem.org/mediawiki/2015/6/6a/Ustc-myo3.png"></p>
-          <p>Raspberry Pi, is the main hardware module that can take photos or videos of interference fringes, then our software analyses them or transmits to your PC. we can see the results on the screen.</p>
-          <div class="divider"></div>
-          <h4 id="Circuit-Elements" class="scrollspy">Circuit Elements</h4>
-          <p><img src="https://static.igem.org/mediawiki/2015/e/e0/Ustc-myo4.png"></p>
-          <ul>
-            <li>Transformer: The responsibility of transformer is to drop voltage 12V　produced by lithium battery to 5V, which is needed to supply cooling fan on Raspberry Pi and laser beam.</li>
-            <li>Breadboard: Many solderlessly electronic circuits tests will be conducted on breadboard.</li>
-            <li>Lithium-polymer battery: Provide power, 12V, 10000mA·h.</li>
-          </ul>
-          <div class="divider"></div>
-          <h4 id="Optical-Path-Elements" class="scrollspy">Optical Path Elements</h4>
-          <p>The schematic figure of optical path is based on Michelson interferometer, to get more principle of optical path in SPRING, please refer to: <a href="http://tower.im">Project-SPRING</a>
-            <br><img src="https://static.igem.org/mediawiki/2015/7/73/Ustc-myo10.png"></p>
-          <p>Here we will introduce elements that are indispensable for interference patterns formation.
-            <br><img src="https://static.igem.org/mediawiki/2015/d/dc/Ustc-myo5.png"></p>
-          <ul>
-            <li>Webcam, containing CCD(charge-coupled device) image sensor, is able to capture picture, mainly for interference pattern capture.</li>
-            <li>Laser, wavelength: 650nm, 5V power supply</li>
-            <li>Expander is to expand the diameter of the laser beam. Two reasons are required to expand beam diameter. On the one hand, a larger diameter of the laser beam could avoid laser focusing on detecting film, preventing the possibility of film burned. On the other hand, laser beam would cover the film for better detection.
-              <br><img src="https://static.igem.org/mediawiki/2015/9/95/Ustc-myo6.png"></li>
-            <li><strong>Beamsplitter combination</strong>, consists of three mirrors, a <strong>50% reflection - 50% transmission beamsplitter(50%(R)/50%(T) in brief)</strong>, a <strong>80% reflection - 20% transmission beamsplitter(80%(R)/20%(T) in brief)</strong> and a <strong>10% reflection- 90% transmission beamsplitter(10%(R)/90%(T) in brief)</strong>. 1)50%(R)/50%(T) is in the front of picture. The reflection light will project to detecting film , while the transmission beamsplitter will continue going to reflection mirror. 2) Then why do we need The 80%(R)/20%(T), along with 10%(R)/90%(T)? Actually these combination is really important to decrease the relatively high intensity reflected from reflector. Much higher intensity from reflector will not fit the formation condition of interference pattern. 80%(R)/20%(T)+10%(R)/90%(T), approximately allowing 18% light approach reflector, are combined together on the bottom of the picture. </li>
-            <li>Reflector, a necessary element for interference pattern formation.</li>
-            <li>Sample trough with film, interacting with reflector to form interference ring.</li>
-          </ul>
-          <div class="divider"></div>
-          <h4 id="Sample-Trough" class="scrollspy">Sample Trough</h4>
-          <p>Our sample trough or sample box is designed based on the most proper size.</p>
-          <p>In the middle lies the empty part, which can be covered by film. </p>
-          <p><img src="https://static.igem.org/mediawiki/2015/6/6e/Ustc-myo7.png" alt="Figure13: Clips"></p>
-          <p>These are our clips, there are two kinds of clips respecitvely used for different shape of film.</p>
-          <p><img src="https://static.igem.org/mediawiki/2015/3/3d/Ustc-myo8.png" alt="Figure14: CAD drawing of clips"></p>
-          <figure>
-            <img src="https://static.igem.org/mediawiki/2015/7/79/Ustc-myo9.png">
-            <figcaption>
-            Figure15: The bases of optical parts
-            </figcaption>
-          </figure>
-          <p>And in order to adjust optical device on the same plain, you may need a plexiglass base to produce. You can produce your own base followed the drawing above.</p>
-          <div class="divider"></div>
-          <h4 id="Film-Preparation" class="scrollspy">Film Preparation</h4>
-          <p>To see more about our ways to find final film through candidate, please refer to <a href="http://tower.im">Modeling-Film Candidate</a>. </p>
-            <p>Our film will be prepared in several processes illustrated below:</p>
-            <p><em>Coating film with polylysine</em></p>
-            <p>Add approximiately 400 ul 20ug/mL polylysine on the film, and store the film at 4 degree celcius for more than 4 hours. After 4 hours, absorb polylysine and then wash the film by PBS buffer. Note: polylysine can be recycled.</p>
-            <p><em>Adhesion Assay</em></p>
-            <p>Add 200 ul bacteria solution on film about 100s. Note: bacteria grown should be in steady state, and you should dilate bacteria and let its OD(600) approximiately reach 0.05</p>
-            <p><em>Operate Optical Path</em></p>
-            <p>Operating optical path within 100s would be highly recommended for users. To get the best images, you should observe and get fringes parallel to y axis of screen as possible.</p>
-            <p><em>Observe and Record</em></p>
-            <p>The measurement period is about 300s. Consequently, we recommend users to take a series of images each 10s during the beginning of 300s. </p>
-          <div class="divider"></div>
-          <h4 id="build-your-electric-circuit" class="scrollspy">Build your electric circuit</h4>
+                            <p>This is our final construction of lac-cheZ <a
-          <p><img src="https://static.igem.org/mediawiki/2015/d/d9/DIANLU.jpg" alt="Figure16: Electric circuit"></p>
+                                    href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a></p>
-            <h5>Prepare RaspberryPi</h5>
-            <p>The Raspberry Pi is a low cost, credit-card sized computer that plugs into a computer monitor or TV, and uses a standard keyboard and mouse. It is a capable little device that enables people of all ages to explore computing, and to learn how to program in languages like Scratch and Python. It’s capable of doing everything you’d expect a desktop computer to do, from browsing the internet and playing high-definition video, to making spreadsheets, word-processing, and playing games.(<a href="http://tower.im">https://www.raspberrypi.org/help/what-is-a-raspberry-pi/</a>)</p>
+                            <p><img src="https://static.igem.org/mediawiki/2015/6/6d/Ustc-t7oprf_lac-chez.jpeg"
-            <p>The module we used is Raspberry-PI 2B, and you can buy from <a href="www.raspberrypi.org">www.raspberrypi.org</a></p>
+                                    alt="Figure 4: plasmid construction of lac-cheZ [BBa_K1593997](http://parts.igem.org/Part:BBa_K1593997) ">
-            <div class="divider"></div>
+                            </p>
-            <h5>First, write the system into a TF card and then put it on your Pi</h5>
-            <p>The system RASPBIAN is recommended. Attention: TF card and SD card are different. TF card also called Micro SD card. You can download the updated system from <a href="www.raspberrypi.org">www.raspberrypi.org</a> </p>
+                            <h4 id="Growth_characteristics" class="scrollspy">Growth characteristics</h4>
-            <p>Then, begin to write. You should prepare a TF card, preferably a high-speed card is recommended above Class4 because the card was a direct impact on the running speed of Raspberry-Pi. </p>
+                            <p>We detected OD600(nm) to identify the effect
-            <p>Get an Installation Tools win32diskimager (under win7), download at <a href="http: //www.onlinedown.net/soft/110173.htm">this site</a></p>
+                                of cheZ (<a href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a>) expressed
-            <p>Connect the TF card to your PC. Unzip the downloaded zip file debian system to give image file.</p>
+                                on <em>E. coli</em> BL21 with time induced by IPTG or not.</p>
-            <p>Extract and run win32diskimager Tools.</p>
-            <p><img src="https://static.igem.org/mediawiki/2015/e/e9/Make_your_own_1-1.jpg" alt=""></p>
+                            <p><img src="https://static.igem.org/mediawiki/2015/d/d6/Ustc-growth.png"
-              <p>Select debian img file, and select the SD drive letter under "Device", then click "Write".</p>
+                                    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">
-              <p><img src="https://static.igem.org/mediawiki/2015/4/42/Make3.jpg" alt=""></p>
+                            </p>
-              <p>Then, you’ll see “Write successful”.</p>
-              <p><img src="https://static.igem.org/mediawiki/2015/7/74/Make4.jpg" alt=""></p>
+                            <p>As the images above illustrated, we found <em>E. coli</em> BL21 wild type has the best
-            <div class="divider"></div>
+                                growth characteristics with a maximal OD600=2.6. Whereas <em>E. coli</em> BL21 with cheZ
-            <h5>Connect and Power on your RaspberryPi</h5>
+                                (<a href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a>) induced by ITPG has
-            <p>You should power your pi with 5 volts using a micro-usb line. Remember to connect your keyboard and mouse to pi first, because you can’t make anything without them.</p>
+                                a relatively low optical density as its OD600 after 9 h is 2.3 compared to the wildtype.
-            <p>Attention: don’t forget to power on the mini fan on the Raspberry-Pi due to too much heat generated.</p>
+                                In the same way, characterization of <em>E. coli</em> BL21 with cheZ (<a
-              <p><img src="https://static.igem.org/mediawiki/2015/a/aa/Make5.jpg" alt=""></p>
+                                        href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a>) not induced by
-              <p>Then connect the display device. </p>
+                                IPTG showed a slightly smaller OD600, as its finally turned to 2.1. The exogenous
-            <p><img src="https://static.igem.org/mediawiki/2015/5/52/Ustc-myo2.png" alt="Figure16: The display module"></p>
+                                protein expression affects the growth of bacteria, which was indicated by slight slower
-            <p>And we need to connect the camera.</p>
+                                grow-up in <em>E. coli</em> containing cheZ plasmid. In total, cheZ doesn't have obvious
-            <p><img src="https://static.igem.org/mediawiki/2015/f/f9/Make6.jpg" alt="Figure17: Webcam"></p>
+                                negative impact on bacteria grown. </p>
-            <p>Finally, get started!</p>
-            <p><img src="https://static.igem.org/mediawiki/2015/e/e0/Make7.jpg" alt="Figure18: Raspberry Pi"></p>
+                            <h4 id="SDS-PAGE" class="scrollspy">SDS-PAGE</h4>
-            <div class="divider"></div>
+                            <p>After induced by IPTG in 1mM about 4 h, cheZ was found in
-            <h5>Get started</h5>
+                                SDS-PAGE gel, compared to the wild type (Top10) and bacteria without IPTG induction.
-            <p>First, you should update your RaspberryPi. Just click the image of monitor. And then enter “sudo apt-get update”.</p>
+                                Through this result, we concluded that cheZ has been successfully expressed with the
-            <p><img src="https://static.igem.org/mediawiki/2015/5/55/Make9.jpg" alt="Figure19: Sudo apt-get update"></p>
+                                IPTG induction.<br><img
-            <p>Then enter “sudo apt-get install luvcview”</p>
+                                        src="https://static.igem.org/mediawiki/2015/7/72/Ustc-chez_expression.png"
-            <p><img src="https://static.igem.org/mediawiki/2015/5/55/Make9.jpg" alt="Figure20: Sudo apt-get install luvcview"></p>
+                                        alt="Figure 6: characterization of cheZ expression by SDS-PAGE"><br>See protocol
-              <p>After installing the software, restart your RaspberryPi and enjoy your journey with SPRING now!</p>
+                                in <a href="http://tower.im">Protocols: SDS-PAGE</a></p>
-            <div class="divider"></div>
-            <h5>How to use Raspberry Pi?</h5>
+                        </div>
-            <p>I. Power on the screen,and then the raspberrypi.Power on the mini fan.</p>
+                    </div>
-            <p>Click this one:</p>
+                    <div class="col hide-on-small-only m3">
-            <p><img src="https://static.igem.org/mediawiki/2015/0/0b/Ustc-perry1.png" alt="Clipboard Image.png"></p>
+                        <div class="toc-wrapper pinned">
-            <p>II. Enter “luvcview” andthen press ”enter”</p>
+                            <ul class="section table-of-contents">
-            <p><img src="https://static.igem.org/mediawiki/2015/9/9b/Ustc-perry2.png" alt="Clipboard Image.png"></p>
+                                <li>
-            <p>III. After enter “luvcview”,Move the table to see the “take a picture!!” Then, click this one. You canclick it for many times.</p>
+                                    <a href="#Genetic_Fragments_Extraction">Genetic Fragments Extraction</a>
-            <p><img src="https://static.igem.org/mediawiki/2015/c/c7/Ustc-perry3.png" alt="Clipboard Image.png"></p>
+                                </li>
-            <p>IV. After get pictures, youcan transfer the files to your computer via WLAN adapter.</p>
+                                <div class="divider"></div>
-            <p>First, you should restart the “networking”.</p>
+                                <li>
-            <p>Enter “sudo /etc/init.d/networking restart”</p>
+                                    <a href="#Growth_characteristics">Growth characteristics</a>
-            <p>You will see this.</p>
+                                </li>
-            <p>You should memory the “bound to XXX.XXX.XX.X” in your mind.</p>
+                                <div class="divider"></div>
-            <p><img src="https://static.igem.org/mediawiki/2015/8/80/Ustc-perry4.png" alt="Clipboard Image.png"></p>
+                                <li>
-            <p>V. Then ,you shouldinstall the software “FileZilla”.</p>
+                                    <a href="#SDS-PAGE">SDS-PAGE</a>
-            <p>Open it ,and enter ”sftp://XXX.XXX.XX.X”</p>
+                                </li>
-            <p>User name is “pi”</p>
+                            </ul>
-            <p>Password is “raspberry”</p>
+                        </div>
-            <p>Normally , the pictures are in /home/pi .</p>
+                    </div>
-            <p>Just drag the file to you computer.</p>
+                </div>
-            <p><img src="https://static.igem.org/mediawiki/2015/f/f7/Ustc-perry5.png" alt="Clipboard Image.png"></p>
+            </div>
-            <div class="divider"></div>
-            <h5>Build your BOX</h5>
-            <p>As is showed in the following picture, there is a clapboard inside the box, above the electric circuit part.</p>
-            <p><img src="https://static.igem.org/mediawiki/2015/b/ba/Make13.jpg" alt="Figure21: Build the box"></p>
-              <p>Oh, remember all parts should be fixed with hot-melt adhesive (hot glue), in order to maintain stability.</p>
-              <p>This is hot glue gun. The gun uses a continuous-duty heating element to melt the plastic glue, which may be pushed through the gun by a mechanical trigger mechanism, or directly by the user. The glue squeezed out of the heated nozzle is initially hot enough to burn and even blister skin. The glue is tacky when hot, and solidifies in a few seconds to one minute. Hot melt adhesives can also be applied by dipping or spraying.</p>
-            <p><img src="https://static.igem.org/mediawiki/2015/3/32/Make18.jpg" alt="Figure22: Hot glue gun"></p>
-              <p>Now, let’s see how to build it.</p>
-              <p>First, bond surfaces of your part and the workbench together. Look at the following picture, pay attention to the white thing. Yes, that’s it.</p>
-            <p><img src="https://static.igem.org/mediawiki/2015/5/5c/Make17.jpg" alt="Figure23: Laser &amp; Expander"></p>
-            <p>Then, stick the optical part with the workbench. Look at the following picture carefully.</p>
-            <p><img src="https://static.igem.org/mediawiki/2015/1/10/Make12.jpg" alt="Figure24: Optical path"></p>
-            <p>And in order to make it easy to adjust the position of the film, we invent a little sliding rail.</p>
-            <p><img src="https://static.igem.org/mediawiki/2015/a/a5/Make11.jpg" alt="Figure25: Sliding rail"></p>
-            <p>As for the clapboard inside the box, we invent a removable device. Actually, it’s just a pair of supporting plates. Notice the left of the following picture.</p>
-            <p><img src="https://static.igem.org/mediawiki/2015/c/cf/Make16.jpg" alt="Figure26: Supporting plates"></p>
-            <p>In this way, we can put the electric circuit part on the supporting plates directly.</p>
-            <p><img src="https://static.igem.org/mediawiki/2015/b/ba/Make13.jpg" alt="Figure27: The inside structure"></p>
-            <p>What's our tool when cutting acrylic?</p>
-            <p><img src="https://static.igem.org/mediawiki/2015/1/19/Make10.jpg" alt="Figure28: Cutting acrylic"></p>
-          </div>
          </div>
-         <div class="col hide-on-small-only m3">
-          <div class="toc-wrapper pinned">
-            <ul class="section table-of-contents">
+         <div id="Adhesion_Assay" class="row">
-              <li>
+            <div class="card hoverable">
-                <a href="#Materials">Materials</a>
+                <div class="col s12 m9">
-              </li>
+                    <div class="card-content">
-              <li>
+                        <h4 id="characterization-of-optimal-conditions-on-polylysine-pll-coated-assay"
-                <a href="#Protection-Guide">Protection Guide</a>
+                            class="scrollspy">
-              </li>
+                            Characterization of Optimal Conditions on Polylysine(PLL) Coated Assay</h4>
-              <li>
-                <a href="#Tools">Tools</a>
+                        <p>To see the details on the mechanism of polylysine adhesion, please refer to <a
-              </li>
+                                href="http://tower.im">Adhesion: Polylysine</a>.</p>
-              <li>
-                <a href="#Display-module">Display module</a>
+                        <p>To get the final PLL-coated protocol, check <a href="http://tower.im">Protocols: PLL
-              </li>
+                            Coated Assay</a> for further information.</p>
-              <li>
-                 <a href="#Raspberry-Pi">Raspberry Pi</a>
+                        <p>Our original characterization of optimal conditions on polylysine(PLL)-coated assay
-              </li>
+                            requires several factors to get, including:</p>
-              <li>
+                        <ol>
-                <a href="#Circuit-Elements">Circuit Elements</a>
+                            <li>Best bacterial developmental interval along with recommended dilution conditions.
-              </li>
+                            </li>
-              <li>
+                            <li>Best PLL-Coated Concentration and Pre-treatment Time.</li>
-                <a href="#Optical-Path-Elements">Optical Path Elements</a>
+                            <li>Best PLL-Coated Time, given full consideration to the completion of bacterial
-              </li>
+                                adhesion time.
-              <li>
+                            </li>
-                <a href="#Sample-Trough">Sample Trough</a>
+                            <li>Best Measurement Interval, which illustrates the possible time for bacterial
-              </li>
+                                response on antibiotics pressure.
-              <li>
+                            </li>
-                <a href="#Film-Preparation">Film Preparation</a>
+                            <li>Possible Determination Interval, which refers to antibiotics response interval.</li>
-              </li>
+                        </ol>
-              <li>
+                        <p>Here we present all the optimistic conditions for users to get the effective results on
-                <a href="#build-your-electric-circuit">Build your electric circuit</a>
+                            antibiotics using our CACCI and SPRING.</p>
-              </li>
-            </ul>
+                        <h4 id="Best_Bacterial_Developmental_Interval_Along_with_Recommended_Dilution_Conditions"
-          </div>
+                            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 that bacteria exploding during <strong>Logarithmic Period(OD:535nm about
+.4~0.5)</strong> are at the most energetic moments with proper bacterial density.
+                            Consequently, we highly recommend user to culture bacteria at Logarithmic Period and
+                            then <strong>dilute bacterial solution about twenty times to fifty times.</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 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
+~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.</p>
+                        <h4 id="Adhesion_Assay_with_PLL_treatment" class="scrollspy">Adhesion Assay with PLL
+                            treatment</h4>
+                        <p>Here we deliver the PLL treatment
+                            results comparing with no PLL treatment assay. During pre-experiment adhesion assay,
+                            PAO1, a 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, which is abbreviated as PLL(-). To observe capability of bacterial adhesion,
+                            we wash observed place with PBS:</p>
+                        <p><img src="https://static.igem.org/mediawiki/2015/c/c3/Control_-1.png"
+                                alt="Figure 1: Control Assay-Without PLL treatment"></p>
+                        <p>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. </p>
+                        <p><em>To get the algorithm of bacterial counting, please refer to <a
+                                href="http://tower.im">Modeling: Bacterial number and movement analysis</a></em>.
+                        </p>
+                        <p><img src="https://static.igem.org/mediawiki/2015/e/e9/PAO1-PLL--.png"
+                                alt="Figure 2: Control Assay-Without PLL treatment2"></p>
+                        <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 twice by PBS.</p>
+                        <p><img src="https://static.igem.org/mediawiki/2015/9/96/Adhesion_Assay.png"
+                                alt="Figure 3: Adhesion Assay-With PLL treatment in 20 ug/mL"></p>
+                        <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. After twice PBS wash, the number of bacteria is relatively stable as well.
+                            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"
+                                alt="Figure 4: Adhesion Assay-With PLL treatment in 20 ug/mL"></p>
+                        <h4 id="Best_PLL-Coated_Time" class="scrollspy">Best PLL-Coated Time</h4>
+                        <p>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 <em>Pseudomonas aeruginosa</em> and HCB1,
+                            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 bacteria increasing during assay.</p>
+                        <p><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">
+                        </p>
+                        <p>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. </p>
+                        <p><img src="https://static.igem.org/mediawiki/2015/e/ee/Developemnt.png"
+                                alt="Figure 6: no antibiotics bacterial number variation through time with PLL"></p>
+                        <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>
+                        <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 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"></p>
+                        <p>After treated with 20ug/mL polylysine, adhesive bacteria number obviously
+                            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 src="https://static.igem.org/mediawiki/2015/d/d8/20150901022.png"
+                                                    alt="图片名称"><br>Simulation 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 Assay Modeling</a></p>
+                        <p>This significant reverse of bacterial adhesion tendency stronly proved that polylysine
+                            has effective adhesive ability for SPRING.</p>
+                        <p>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.</p>
+                        <p>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.</p>
+                        <p>Consequently, we recommended that PLL treatment after 5 min would be a promising set for
+                            SPRING to output stable data.</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 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.</p>
+                        <p>The measurement assay is illustrated as following:</p>
+                        <p><em>PLL(-)</em></p>
+                        <table class="striped">
+                            <thead>
+                            <tr>
+                                <th>Antibiotics Concentration(ug/mL)</th>
+                                <th>Incubation Time(s)</th>
+                            </tr>
+                            </thead>
+                            <tbody>
+                            <tr>
+                                <td>0</td>
+                                <td>0</td>
+                            </tr>
+                            <tr>
+                                <td>0</td>
+                                <td>60</td>
+                            </tr>
+                            <tr>
+                                <td>0</td>
+                                <td>120</td>
+                            </tr>
+                            <tr>
+                                <td>0</td>
+                                <td>180</td>
+                            </tr>
+                            <tr>
+                                <td>0</td>
+                                <td>240</td>
+                            </tr>
+                            <tr>
+                                <td>0</td>
+                                <td>300</td>
+                            </tr>
+                            <tr>
+                                <td>1</td>
+                                <td>0</td>
+                            </tr>
+                            <tr>
+                                <td>1</td>
+                                <td>60</td>
+                            </tr>
+                            <tr>
+                                <td>1</td>
+                                <td>300</td>
+                            </tr>
+                            </tbody>
+                        </table>
+                        <p><em>PLL(+,20)</em></p>
+                        <table  class="striped">
+                            <thead>
+                            <tr>
+                                <th>Antibiotics Concentration(ug/mL)</th>
+                                <th>Incubation Time(s)</th>
+                            </tr>
+                            </thead>
+                            <tbody>
+                            <tr>
+                                <td>0</td>
+                                <td>0</td>
+                            </tr>
+                            <tr>
+                                <td>0</td>
+                                <td>60</td>
+                            </tr>
+                            <tr>
+                                <td>0</td>
+                                <td>120</td>
+                            </tr>
+                            <tr>
+                                <td>0</td>
+                                <td>180</td>
+                            </tr>
+                            <tr>
+                                <td>0</td>
+                                <td>240</td>
+                            </tr>
+                            <tr>
+                                <td>0</td>
+                                <td>300</td>
+                            </tr>
+                            <tr>
+                                <td>0.1</td>
+                                <td>0</td>
+                            </tr>
+                            <tr>
+                                <td>0.1</td>
+                                <td>60</td>
+                            </tr>
+                            <tr>
+                                <td>0.1</td>
+                                <td>300</td>
+                            </tr>
+                            <tr>
+                                <td>0.5</td>
+                                <td>0</td>
+                            </tr>
+                            <tr>
+                                <td>0.5</td>
+                                <td>60</td>
+                            </tr>
+                            <tr>
+                                <td>0.5</td>
+                                <td>300</td>
+                            </tr>
+                            <tr>
+                                <td>1</td>
+                                <td>0</td>
+                            </tr>
+                            <tr>
+                                <td>1</td>
+                                <td>60</td>
+                            </tr>
+                            <tr>
+                                <td>1</td>
+                                <td>300</td>
+                            </tr>
+                            </tbody>
+                        </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 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, 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>
+                        <p>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.</p>
+                        <p>As for bacteria treated with 20 ug/mL PLL, different pattern is observed:</p>
+                        <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 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 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>
+                        <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>
+                        <p>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.</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>
-    </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>
+                        <h2 id="modeling-guide-us-choose-the-candidates" class="scrollspy">Modeling guide us choose the
+                            candidates</h2>
+                        <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>
+                        <h2 id="processing-film" class="scrollspy">Processing Film</h2>
+                        <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>
+                        <h2 id="film-i" class="scrollspy">Film I</h2>
+                        <p><strong>Low Pressure Polyethylene</strong><br><img
+                                src="https://static.igem.org/mediawiki/2015/e/ee/Ustc-filmcandid1.png"
+                                alt="Figure 1: Film I, polyethylene"></p>
+                        <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>
+                        <h2 id="film-ii" class="scrollspy">Film II</h2>
+                        <p><strong>Rubbers-Condom</strong><br><img
+                                src="https://static.igem.org/mediawiki/2015/b/b7/Ustc-filmcandid3.jpeg"
+                                alt="Figure 2: Film II, condom"></p>
+                        <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>
+                        <h2 id="film-iii" class="scrollspy">Film III</h2>
+                        <p><strong>Cover slip</strong><br><img
+                                src="https://static.igem.org/mediawiki/2015/e/ef/Ustc-filmcandid2.png"
+                                alt="Figure 3: Film III, Cover slip"></p>
+                        <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><img
+                                    src="https://static.igem.org/mediawiki/2015/b/b8/20150918067.jpg"
+                                    alt="Figure 4 :Relation between fringes changes and antibiotics concentration"></p>
+                        <p>Then we want to develop a calibration with three point.<br><img
+                                src="https://static.igem.org/mediawiki/2015/e/e4/20150918066.jpg"
+                                alt="Figure 5: Experimental results of calibration"><br>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>
-  </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