Difference between revisions of "Team:ECUST/Lysin"

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<p>In order to test the autolysis effect of Lysin, we constructed the vector pET28a-Lysin. </p>
 
<p>In order to test the autolysis effect of Lysin, we constructed the vector pET28a-Lysin. </p>
 
<figure>
 
<figure>
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<figure class="makeresponsive" style="width: 50%;margin-left:22%;margin-right:30%;">
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<figure class="makeresponsive" style="width: 50%;margin-left:24%;margin-right:30%;">
 
<img src="https://static.igem.org/mediawiki/2018/2/22/T--ECUST--result--lysin1.jpg" class="z1">
 
<img src="https://static.igem.org/mediawiki/2018/2/22/T--ECUST--result--lysin1.jpg" class="z1">
 
<figcaption><b>Figure 1. The vector pET-28a Vector is cut by NcoI and BamHI. Sequence of lysin is sourced from biobrick BBa_K117000. Gene sequence of lysin is amplified by PCR and is ligated with linearized vector by Ezmax.</b></figcaption>
 
<figcaption><b>Figure 1. The vector pET-28a Vector is cut by NcoI and BamHI. Sequence of lysin is sourced from biobrick BBa_K117000. Gene sequence of lysin is amplified by PCR and is ligated with linearized vector by Ezmax.</b></figcaption>
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<p>The plasmid was transformed to E. coli BL21 and cultured at 37 °C for 12h. positive monoclonal bacteria were cultured and verified by PCR. </p>
 
<p>The plasmid was transformed to E. coli BL21 and cultured at 37 °C for 12h. positive monoclonal bacteria were cultured and verified by PCR. </p>
 
<figure>
 
<figure>
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<figure class="makeresponsive" style="width: 50%;margin-left:22%;margin-right:30%;">
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<figure class="makeresponsive" style="width: 50%;margin-left:24%;margin-right:30%;">
 
<img src="https://static.igem.org/mediawiki/2018/2/28/T--ECUST--result--lysin2.jpg" class="z2">
 
<img src="https://static.igem.org/mediawiki/2018/2/28/T--ECUST--result--lysin2.jpg" class="z2">
 
<figcaption><b>Figure 2. 1% Agarose Gel Electrophoresis of PCR, which shows that our vector was successfully constructed.</b></figcaption>
 
<figcaption><b>Figure 2. 1% Agarose Gel Electrophoresis of PCR, which shows that our vector was successfully constructed.</b></figcaption>
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<p>Recombinant bacteria is cultured by LB medium adding with 0.1% kanamycin till logarithmic phase and induced by IPTG. Then culture solution is transferred into 96-well microtiter plates and measured the light absorption of 600 nm. </p>
 
<p>Recombinant bacteria is cultured by LB medium adding with 0.1% kanamycin till logarithmic phase and induced by IPTG. Then culture solution is transferred into 96-well microtiter plates and measured the light absorption of 600 nm. </p>
 
<figure>
 
<figure>
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<figure class="makeresponsive" style="width: 50%;margin-left:22%;margin-right:30%;">
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<figure class="makeresponsive" style="width: 50%;margin-left:24%;margin-right:30%;">
 
<img src="https://static.igem.org/mediawiki/2018/c/ca/T--ECUST--result--lysin3.jpg" class="z3">
 
<img src="https://static.igem.org/mediawiki/2018/c/ca/T--ECUST--result--lysin3.jpg" class="z3">
 
<figcaption><b>Figure 3. Cell growth curve with or without IPTG induction. E. coli without IPTG induction grew normally while the density of E.coli decreased after addition of IPTG.</b></figcaption>
 
<figcaption><b>Figure 3. Cell growth curve with or without IPTG induction. E. coli without IPTG induction grew normally while the density of E.coli decreased after addition of IPTG.</b></figcaption>
 
</figure>
 
</figure>
 
<figure>
 
<figure>
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<figure class="makeresponsive" style="width: 50%;margin-left:22%;margin-right:30%;">
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<figure class="makeresponsive" style="width: 50%;margin-left:24%;margin-right:30%;">
 
<img src="https://static.igem.org/mediawiki/2018/b/b4/T--ECUST--result--lysin4.jpg" class="z4">
 
<img src="https://static.igem.org/mediawiki/2018/b/b4/T--ECUST--result--lysin4.jpg" class="z4">
 
<figcaption><b>Figure 4. The images are from scanning electron microscopes. (a)Before induction.(b)After induction. There are holes in the plasma membrane after induction, which can prove that lysin expresses successfully.</b></figcaption>
 
<figcaption><b>Figure 4. The images are from scanning electron microscopes. (a)Before induction.(b)After induction. There are holes in the plasma membrane after induction, which can prove that lysin expresses successfully.</b></figcaption>

Revision as of 22:12, 17 October 2018

LYSIN

Design

no

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

In order to test the autolysis effect of Lysin, we constructed the vector pET28a-Lysin.

Figure 1. The vector pET-28a Vector is cut by NcoI and BamHI. Sequence of lysin is sourced from biobrick BBa_K117000. Gene sequence of lysin is amplified by PCR and is ligated with linearized vector by Ezmax.

The plasmid was transformed to E. coli BL21 and cultured at 37 °C for 12h. positive monoclonal bacteria were cultured and verified by PCR.

Figure 2. 1% Agarose Gel Electrophoresis of PCR, which shows that our vector was successfully constructed.

In order to verify whether the cell expression of Lysin can perforate on the plasma membrane, we test the growth curve of recombinant E.coli and check them under the scanning electron microscope.

Recombinant bacteria is cultured by LB medium adding with 0.1% kanamycin till logarithmic phase and induced by IPTG. Then culture solution is transferred into 96-well microtiter plates and measured the light absorption of 600 nm.

Figure 3. Cell growth curve with or without IPTG induction. E. coli without IPTG induction grew normally while the density of E.coli decreased after addition of IPTG.
Figure 4. The images are from scanning electron microscopes. (a)Before induction.(b)After induction. There are holes in the plasma membrane after induction, which can prove that lysin expresses successfully.