Difference between revisions of "Team:USTC/Parts"

Revision as of 01:58, 19 September 2015

Overview
Chemotaxis Modification
Permeability Improvement
Adhesion Assay
Parts

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.
Antibiotics Concentration Detection Demo: This section includes our initial test on our system, how we define our final methods and how we establshed protocols for users.
Calibration

Want to get them now? Click their title directly!

Growth characteristics
We detected OD600(nm) to identify the effect of cheZ expression on E. coli through time.
图片名称

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.
图片名称
See protocol in Protocols: SDS-PAGE

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

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.

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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.
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After treated with 20ug/mL polylysine, adhesive bacteria number obviously increased:
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Modeling on adhesion process strictly demonstrated the adhesion assay fits Langmuir absorption isoform.

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

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

<groupparts>iGEM015 Example</groupparts>

@@ Line 9: / Line 9: @@
          <ul class="tabs tabs-wrapper transparent">
              <li class="tab col l2 m2 s2">
-                 <a href="#Basic_Parts" class="blue-text active waves-effect waves-light">Basic Parts</a>
+                 <a href="#Overview" class="blue-text active waves-effect waves-light">Overview</a>
              </li>
              <li class="tab col l2 m2 s2">
-                 <a href="#Composite_Parts" class="blue-text waves-effect waves-light">Composite Parts</a>
+                 <a href="#Chemotaxis_Modification" class="blue-text waves-effect waves-light">Chemotaxis
+                    Modification</a>
+            </li>
+            <li class="tab col l2 m2 s2">
+                <a href="#Permeability_Improvement"
+                   class="blue-text waves-effect waves-light">Permeability Improvement</a>
+            </li>
+            <li class="tab col l2 m2 s2">
+                <a href="#Adhesion_Assay" class="blue-text waves-effect waves-light">Adhesion Assay</a>
              </li>
              <li class="tab col l2 m2 s2">
                  <a href="#Parts" class="blue-text waves-effect waves-light">Parts</a>
              </li>
          </ul>
      </div>
@@ Line 24: / Line 31: @@
      <li><a href="https://2015.igem.org/Team:USTC" class="waves-effect waves-light">Home</a></li>
      <li><a href="https://2015.igem.org/Team:USTC/Description" class="waves-effect waves-light">Project</a></li>
+    <li><a href="https://2015.igem.org/Team:USTC/Modeling" class="waves-effect waves-light">Modeling</a></li>
      <li><a href="https://2015.igem.org/Team:USTC/Achievements" class="waves-effect waves-light">Achivements</a></li>
      <li><a href="https://2015.igem.org/Team:USTC/Tutorials" class="waves-effect waves-light">Tutorials</a></li>
@@ Line 36: / Line 44: @@
 <div class="row">
      <div class="col offset-m1 offset-l2 s12 m10 l8">
-         <div id="Basic_Parts" class="row">
+         <div id="Overview" class="row">
              <div class="card hoverable">
-                 <div class="col s12 m9">
+                 <div class="col s12">
                      <div class="card-content">
-                         <p>This year, we choose several parts to conduct our projects. The main functions of the
+                         <p>That is so amazing! We finally made NDM this year! The results page will deliver the fresh
-                             function we use are as follow:</p>
+                             results from us. Our results include these sections:</p>
-                         <ol>
+                         <ul>
-                             <li>Promoters.</li>
+                             <li><p>Permeability Improvement: In this section, we will demonstrate our synthetic
-                            <li>Proteins to improve the bactial permeability.</li>
+                                 bioloical base constrcuction, characterization of permeability improvement. This is the
-                            <li>Porins.</li>
+                                 fundation of CACCI construction, as well as NDM.</p>
-                            <li>Proteins to improve the mobile of bacteria.</li>
+                             </li>
-                            <li>Sense system.</li>
+                             <li><p>Chemotaxis Modification: This section introduces results of chemotaxis
-                        </ol>
+                                 engineering.</p>
-                        <table class="striped">
+                             </li>
-                            <thead>
+                             <li><p>Adhesion Assay: This section explicitly explain adhesion methods, adhesion dynamics
-                            <tr>
+                                 and adhesion protocols recommended for all users, which is an important part of our
-                                <th>Code Name</th>
+                                 results.</p>
-                                <th>Part Name</th>
+                             </li>
-                                <th>Function</th>
+                             <li><p>Antibiotics Concentration Detection Demo: This section includes our initial test on
-                            </tr>
+                                 our system, how we define our final methods and how we establshed protocols for
-                            </thead>
+                                 users.</p>
-                            <tbody>
+                             </li>
-                            <tr>
+                             <li><p>Calibration</p>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593207">BBa_K1593207</a></td>
+                            </li>
-                                 <td>micF</td>
+                        </ul>
-                                <td>Promoter sensing sulfoamid</td>
+                        <p>Want to get them now? Click their title directly!</p>
-                            </tr>
-                            <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593208">BBa_K1593208</a></td>
-                                <td>SoxS</td>
-                                <td>Promoter sensing tetracycline</td>
-                            </tr>
-                            <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593209">BBa_K1593209</a></td>
-                                <td>OprF</td>
-                                <td>The major porin of Pseudomonas aeruginosa allowing diffusion of polysaccharides in a
-                                    range of 2000 to 3000 Da.
-                                </td>
-                            </tr>
-                            <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593666">BBa_K1593666</a></td>
-                                <td>SCVE</td>
-                                <td>SARS Coronavirus Envelope Protein, improving the bactial permeability.</td>
-                            </tr>
-                            <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593998">BBa_K1593998</a></td>
-                                <td>cheZ</td>
-                                <td>Chemotactic protein. When it is overexpressed, the mobility of bacteria will be
-                                    improved.
-                                 </td>
-                            </tr>
-                            <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a></td>
-                                <td>T7-RBS-SCVE</td>
-                                <td>Circuit containing strong expression of SCVE regulated by T7, which will
-                                    significantly improve bacterial permeability.
-                                </td>
-                             </tr>
-                             <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a></td>
-                                 <td>T7-RBS-OprF</td>
-                                <td>Using T7 strong promoter to express OprF, derived from Pseudomonus areuginosa, would
-                                    strongly improve the bacterial permeability.
-                                </td>
-                             </tr>
-                             <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a></td>
-                                 <td>R0010-RBS-cheZ</td>
-                                 <td>Used to coding a chemotactci protein, cheZ, downstream of lac promotor.</td>
-                             </tr>
-                             <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593211">BBa_K1593211</a></td>
-                                 <td>micF-RBS-GFP</td>
-                                 <td>A basic part on antibiotic substance sensing-reporting system.</td>
-                             </tr>
-                             <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593001">BBa_K1593001</a></td>
-                                <td>COMPX</td>
-                                <td>Express an outmembrane protein with a point mutation</td>
-                            </tr>
-                            </tbody>
-                        </table>
                      </div>
@@ Line 126: / Line 78: @@
-         <div id="Composite_Parts" class="row">
+         <div id="Chemotaxis_Modification" class="row">
              <div class="card hoverable">
-                 <div class="col s12 m9">
+                 <div class="row">
-                     <div class="card-content">
+                     <div class="col s12">
-                         <p>A list of composit parts USTC iGEM 2015 have submitted to the Registry.The main functions of
+                         <div class="card-content">
-                            these composite parts are as following:</p>
+                             <p><strong>Growth characteristics</strong><br>We detected OD600(nm) to identify the effect
-                        <ol>
+                                 of cheZ expression on <em>E. coli</em> through time.<br><img
-                            <li>To improve the permeability of bacteria;</li>
+                                        src="https://static.igem.org/mediawiki/2015/d/d6/Ustc-growth.png" alt="图片名称"></p>
-                            <li>To modify the chemotaxis of bacteria;</li>
-                            <li>To modify outmembrane protein;</li>
-                            <li>To test the response of promtor.</li>
-                        </ol>
-                        <table class="striped">
-                             <thead>
-                            <tr>
-                                <th>Code Name</th>
-                                <th>Parts Name</th>
-                                <th>Type</th>
-                                <th>Function</th>
-                            </tr>
-                            </thead>
-                            <tbody>
-                            <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593667">BBa_K1593667</a></td>
-                                <td>T7-RBS-SCVE</td>
-                                <td>Translational_Unit</td>
-                                <td>overexpress SCVE to improve the permeability of bacterial</td>
-                            </tr>
-                            <tr>
-                                 <td><a href="http://parts.igem.org/Part:BBa_K1593210">BBa_K1593210</a></td>
-                                <td>T7-RBS-OprF</td>
-                                <td>Coding</td>
-                                <td>overexpress OprF to improve the permeability of bacterial</td>
-                            </tr>
-                            <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593997">BBa_K1593997</a></td>
-                                <td>R0010-RBS-cheZ</td>
-                                <td>Coding</td>
-                                <td>overexpress cheZ under the control of lac to improve the chemotaxis of bacterial
-                                </td>
-                            </tr>
-                            <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593211">BBa_K1593211</a></td>
-                                <td>micF-RBS-GFP</td>
-                                <td>Translational_Unit</td>
-                                <td>test the response of micF to antibiotics using the GFP report system</td>
-                            </tr>
-                            <tr>
-                                <td>
-                                    <a href="http://parts.igem.org/cgi/partsdb/part_info.cgi?part_id=38524">BBa_K1593888</a>
-                                </td>
-                                <td>tRNA&amp;tRNA Synthetase</td>
-                                <td>Translational_Unit</td>
-                                <td>express a rare amino acid,pAzF</td>
-                            </tr>
-                            <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593002">BBa_K1593002</a></td>
-                                <td>tRNA complex with Compx</td>
-                                <td>Device</td>
-                                <td>orthogonal system to express Compx</td>
-                            </tr>
-                            <tr>
-                                <td><a href="http://parts.igem.org/Part:BBa_K1593000">BBa_K1593000</a></td>
-                                <td>lac-compx with RFP</td>
-                                <td>Device</td>
-                                <td>overexpress compx and detect its quantity by GFP</td>
-                            </tr>
-                            </tbody>
-                        </table>
+                            <p>The exogenous protein expression affects the growth of bacteria, which was indicated by
+                                slight slower grow-up in <em>E. coli</em> containing cheZ plasmid. In total, cheZ
+                                doesn't have obvious negative impact on bacteria grown. </p>
+                            <p><strong>SDS-PAGE</strong><br>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.<br><img
+                                        src="https://static.igem.org/mediawiki/2015/7/72/Ustc-chez_expression.png" alt="图片名称"><br>See
+                                protocol in <a href="http://tower.im">Protocols: SDS-PAGE</a></p>
+                        </div>
                      </div>
                  </div>
+            </div>
+        </div>
+        <div id="Permeability_Improvement" class="row">
+            <div class="card hoverable">
+                <div class="row">
+                    <div class="col s12">
+                        <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>
+                                <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="divider"></div>
+                                <li>
+                                    <a href="#bibliography">Bibliography</a>
+                                </li>
+                            </ul>
+                        </div>
+                    </div>
+                </div>
              </div>
          </div>
+        <div id="Adhesion_Assay" class="row">
+            <div class="card hoverable">
+                <div class="col s12">
+                    <div class="card-content">
+                        <h3 id="characterization-of-optimal-conditions-on-polylysine-pll-coated-assay" class="scrollspy">
+                            Characterization of Optimal Conditions on Polylysine(PLL) Coated Assay</h3>
+                            <p>To see the details on the mechanism of polylysine adhesion, please refer to <a
+                                    href="http://tower.im">Adhesion: Polylysine</a>.</p>
+                            <p>To get the final PLL-coated protocol, check <a href="http://tower.im">Protocols: PLL
+                                Coated Assay</a> for further information.</p>
+                            <p>Our original characterization of optimal conditions on polylysine(PLL)-coated assay
+                                requires several factors to get, including:</p>
+                            <ol>
+                                <li>Best bacterial developmental interval along with recommended dilution conditions.
+                                </li>
+                                <li>Best PLL-Coated Concentration and Pre-treatment Time.</li>
+                                <li>Best PLL-Coated Time, given full consideration to the completion of bacterial
+                                    adhesion time.
+                                </li>
+                                <li>Best Measurement Interval, which illustrates the possible time for bacterial
+                                    response on antibiotics pressure.
+                                </li>
+                                <li>Possible Determination Interval, which refers to antibiotics response interval.</li>
+                            </ol>
+                            <p>Here we present all the optimistic conditions for users to get the effective results on
+                                antibiotics using our CACCI and SPRING.</p>
+                            <h3 id="Best_Bacterial_Developmental_Interval_Along_with_Recommended_Dilution_Conditions" class="scrollspy">Best Bacterial Developmental Interval Along with Recommended Dilution
+                                Conditions</h3>
+                            <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>
+                            <h3 id="Best_PLL-Coated_Concentration_and_Pre-treatment_Time" class="scrollspy">Best PLL-Coated Concentration and Pre-treatment Time.</h3>
+                            <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>
+                            <h3 id="Adhesion_Assay_with_PLL_treatment" class="scrollspy">Adhesion Assay with PLL treatment</h3>
+                            <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>
+                            <h3 id="Best_PLL-Coated_Time" class="scrollspy">Best PLL-Coated Time</h3>
+                            <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>
+                            <h3 id="Best_Measurement_Interval" class="scrollspy">Best Measurement Interval</h3>
+                            <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>
+                                <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>
+                                <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 id="Parts" class="row">
@@ Line 206: / Line 763: @@
                  <div class="col s12 m9">
                      <div class="card-content">
-                         <groupparts>iGEM015 Example</groupparts>
+                         </html>
+<groupparts>iGEM015 Example</groupparts>
+<html>
+                    </div>
                      </div>
                  </div>
              </div>
-        </div>
+        </div>
      </div>

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