Difference between revisions of "Team:ECUST/Light-on Suicide"

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<h1 class="box-heading">Design</h1>
 
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<p>“Human Practices is the study of how your work affects the world, and how the world affects the work.” (Peter Carr, Director of Judging) is the comment which gives a clear definition of all Human Practices activities. Therefore, we tried to answer two questions: how can synthetic biology affect the world? And in which way can the world affect Iron Guardian? To answer the former one, we chose to collaborate with different teams and thought the issues we are working on vary from team to team, we all wish and try to utilize what we learn to solve practical problems and make the world a better one. What’s more, we integrated the social influence into our project to answer the latter question. With communication, economic benefit and responsibility as our important focus, we interviewed professors and employees in related industry, and showcased the great economic benefit of our project. By distributing questionnaire and analyzing the data acquired, we managed to explain and improve our project according to the public concerns and suggestions. The new concept of “3+ net” raised by ECUST will also help all teams to create, to reflect, and to improve.</p>
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<p>The leakage of engineered <i>E. coli</i> from the pipeline system into the natural environment could be of potential risk, and to prevent that from happening, we need to introduce a suicide system. Considering that the pipeline system is a semi-closed, no-light environment, we used light-on system to express nucleases to kill engineered <i>E.coli</i> under conditions with light. The light-on system is realized by the light-sensitive protein LEVI. In the absence of light, the LEVI protein is a protein monomer, which is often expressed in cells. When exposed to certain wavelengths of light, LEVI will dimerize and bind to specific sequences to inhibit gene expression. Therefore, similar to the principle of the iron reversal system, we need to introduce the cI gene, and cI will inhibit the promoter Pλ (R-O12). Therefore, when light is present, the expression of the nucleic acid hydrolysis gene can be initiated.</p>
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<figcaption><b>Figure 1</b></figcaption>
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<h1 class="box-heading">Construct</h1>
 
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<p>In order to test the biofilm removal effect of DSPB, we constructed the vector pET28a-DSPB</p>
 
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<p>The plasmid was transformed into E. coli BL21, cultured at 37 °C for 12 h, and the plasmid was extracted and verified by PCR.</p>
 
  
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<p>First, insert the mCherry reporter gene into the vector pLEVI0n to characterize the effect of light control system. Plasmid pLEVIOn is from Professor Yi Yang. </p>
 
<p>First, insert the mCherry reporter gene into the vector pLEVI0n to characterize the effect of light control system. Plasmid pLEVIOn is from Professor Yi Yang. </p>
 
<p>The plasmids are transformed to E.coil 3J1.Positive bacteria is cultured in LB culture medium adding with 0.1% streptomycin overnight, then 1% inoculum of culture solution is operated. Cells with light illumination all the time, with light illumination until logarithmic phase, with light illumination until late period of logarithmic phase and cells in dark are measured fluorescence intensity. Wavelength of exciting light is 587nm, and wavelength of emitted light is 610nm. </p>
 
<p>The plasmids are transformed to E.coil 3J1.Positive bacteria is cultured in LB culture medium adding with 0.1% streptomycin overnight, then 1% inoculum of culture solution is operated. Cells with light illumination all the time, with light illumination until logarithmic phase, with light illumination until late period of logarithmic phase and cells in dark are measured fluorescence intensity. Wavelength of exciting light is 587nm, and wavelength of emitted light is 610nm. </p>
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<p>The period starting light illumination doesn’t affect on the expression of mcherry, so no matter which growth phase bacteria is, the system can response. </p>
 
<p>The period starting light illumination doesn’t affect on the expression of mcherry, so no matter which growth phase bacteria is, the system can response. </p>
 
<p>Then we insert MNase to vector pLEVIOn. Vector is cut by Pst1 and Spe1, Mnase is from biobrick BBa_K902019.  Exogenous gene is amplified by PCR and then linearized vector and gene fragment is ligated with Ezmax. The plasmids are transformed to E.coil 3J1.Positive bacteria is cultured in LB culture medium adding with 0.1% streptomycin overnight, then 1% inoculum of culture solution is operated. Cells were cultured in dark,then was extracted and verified by PCR. </p>
 
<p>Then we insert MNase to vector pLEVIOn. Vector is cut by Pst1 and Spe1, Mnase is from biobrick BBa_K902019.  Exogenous gene is amplified by PCR and then linearized vector and gene fragment is ligated with Ezmax. The plasmids are transformed to E.coil 3J1.Positive bacteria is cultured in LB culture medium adding with 0.1% streptomycin overnight, then 1% inoculum of culture solution is operated. Cells were cultured in dark,then was extracted and verified by PCR. </p>
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<img src="https://static.igem.org/mediawiki/2018/f/fa/T--ECUST--result--IIGHT_ON_2.jpg" class="z2">
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<figcaption><b>Figure 2. 1% Agarose Gel Electrophoresis of MNase </b></figcaption>
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<figcaption><b>Figure 3: <i>E.coli</i> growth curve under different light conditions. It can be seen that cell growth can be inhibited after light illumination.</b></figcaption>
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<h1 class="box-heading">Reference</h1>
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<p>1. Cell Research (2016) 26:854-857. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Xianjun Chen, Renmei Liu, Zhengcai Ma, Xiaopei Xu, Haoqian Zhang, Jianhe Xu, Qi Ouyang, Yi Yang</p>
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Latest revision as of 00:10, 18 October 2018

Light On Suicide

Design

The leakage of engineered E. coli from the pipeline system into the natural environment could be of potential risk, and to prevent that from happening, we need to introduce a suicide system. Considering that the pipeline system is a semi-closed, no-light environment, we used light-on system to express nucleases to kill engineered E.coli under conditions with light. The light-on system is realized by the light-sensitive protein LEVI. In the absence of light, the LEVI protein is a protein monomer, which is often expressed in cells. When exposed to certain wavelengths of light, LEVI will dimerize and bind to specific sequences to inhibit gene expression. Therefore, similar to the principle of the iron reversal system, we need to introduce the cI gene, and cI will inhibit the promoter Pλ (R-O12). Therefore, when light is present, the expression of the nucleic acid hydrolysis gene can be initiated.

Figure 1

Result

First, insert the mCherry reporter gene into the vector pLEVI0n to characterize the effect of light control system. Plasmid pLEVIOn is from Professor Yi Yang.

The plasmids are transformed to E.coil 3J1.Positive bacteria is cultured in LB culture medium adding with 0.1% streptomycin overnight, then 1% inoculum of culture solution is operated. Cells with light illumination all the time, with light illumination until logarithmic phase, with light illumination until late period of logarithmic phase and cells in dark are measured fluorescence intensity. Wavelength of exciting light is 587nm, and wavelength of emitted light is 610nm.

The period starting light illumination doesn’t affect on the expression of mcherry, so no matter which growth phase bacteria is, the system can response.

Then we insert MNase to vector pLEVIOn. Vector is cut by Pst1 and Spe1, Mnase is from biobrick BBa_K902019. Exogenous gene is amplified by PCR and then linearized vector and gene fragment is ligated with Ezmax. The plasmids are transformed to E.coil 3J1.Positive bacteria is cultured in LB culture medium adding with 0.1% streptomycin overnight, then 1% inoculum of culture solution is operated. Cells were cultured in dark,then was extracted and verified by PCR.

Figure 2. 1% Agarose Gel Electrophoresis of MNase
Figure 3: E.coli growth curve under different light conditions. It can be seen that cell growth can be inhibited after light illumination.

Reference

1. Cell Research (2016) 26:854-857. An extraordinary stringent and sensitive light-switchable gene expression system for bacterial cells. Xianjun Chen, Renmei Liu, Zhengcai Ma, Xiaopei Xu, Haoqian Zhang, Jianhe Xu, Qi Ouyang, Yi Yang