Difference between revisions of "Team:ECUST/Light-on Suicide"
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<p>In order to prevent the leakage of engineered E. coli from the pipeline system into the natural environment and create potential biosafety problems, we need to introduce a suicide system. Considering that the piping system is a semi-closed, no-light environment, we used light-on system to express nucleases to make engineered E.coli to die under light conditions. 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 illuminated under 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> | <p>In order to prevent the leakage of engineered E. coli from the pipeline system into the natural environment and create potential biosafety problems, we need to introduce a suicide system. Considering that the piping system is a semi-closed, no-light environment, we used light-on system to express nucleases to make engineered E.coli to die under light conditions. 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 illuminated under 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> | ||
<figure> | <figure> | ||
| − | <figure class="makeresponsive" style="width: 50%;"> | + | <figure class="makeresponsive" style="width: 50%;margin-left:25%;"> |
<img src="https://static.igem.org/mediawiki/2018/f/f4/T--ECUST--lighton.png" class="z3"> | <img src="https://static.igem.org/mediawiki/2018/f/f4/T--ECUST--lighton.png" class="z3"> | ||
<figcaption><b>Figure 1</b></figcaption> | <figcaption><b>Figure 1</b></figcaption> | ||
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<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> | ||
<figure> | <figure> | ||
| − | <figure class="makeresponsive" style="width: 50%;"> | + | <figure class="makeresponsive" style="width: 50%;margin-left:22%;"> |
<img src="https://static.igem.org/mediawiki/2018/f/fa/T--ECUST--result--IIGHT_ON_2.jpg" class="z2"> | <img src="https://static.igem.org/mediawiki/2018/f/fa/T--ECUST--result--IIGHT_ON_2.jpg" class="z2"> | ||
<figcaption><b>Figure 2. 1% Agarose Gel Electrophoresis of MNase </b></figcaption> | <figcaption><b>Figure 2. 1% Agarose Gel Electrophoresis of MNase </b></figcaption> | ||
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<figure> | <figure> | ||
| − | <figure class="makeresponsive" style="width: 50%;"> | + | <figure class="makeresponsive" style="width: 50%;margin-left:22%;"> |
<img src="https://static.igem.org/mediawiki/2018/e/ef/T--ECUST--result--IIGHT_ON_3.jpg" class="z3"> | <img src="https://static.igem.org/mediawiki/2018/e/ef/T--ECUST--result--IIGHT_ON_3.jpg" class="z3"> | ||
<figcaption><b>Figure 3: E.coli growth curve under different light conditions. It can be seen that cell growth can be inhibited after light illumination.</b></figcaption> | <figcaption><b>Figure 3: E.coli growth curve under different light conditions. It can be seen that cell growth can be inhibited after light illumination.</b></figcaption> | ||
Revision as of 22:26, 17 October 2018
Design
In order to prevent the leakage of engineered E. coli from the pipeline system into the natural environment and create potential biosafety problems, we need to introduce a suicide system. Considering that the piping system is a semi-closed, no-light environment, we used light-on system to express nucleases to make engineered E.coli to die under light conditions. 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 illuminated under 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.
Construct
In order to test the biofilm removal effect of DSPB, we constructed the vector pET28a-DSPB
The plasmid was transformed into E. coli BL21, cultured at 37 °C for 12 h, and the plasmid was extracted and verified by PCR.
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.
