Difference between revisions of "Team:ECUST/Lysin"

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     <div id="bannerquote">Overview</div>
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     <div id="bannerquote">Lysin</div>
 
      
 
      
 
 
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<h1 class="box-heading">Design</h1>
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<h1 class="box-heading">Description and 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>Lysis gene codes for the colicin-producing bacteria and then activates the expression of colicin. Colicin is a type of bacteriocin produced by and toxic to some strains of <i>Escherichia coli</i>. Colicins are released into the environment to reduce competition from other bacterial strains. Colicins bind to outer membrane receptors, using them to translocate to the cytoplasm or cytoplasmic membrane, where they exert their cytotoxic effect, including depolarisation of the cytoplasmic membrane, DNase activity, RNase activity, or inhibition of murein synthesis. colicins contain at least three domains: an N-terminal translocation domain responsible for movement across the outer membrane and periplasmic space; a central domain responsible for receptor recognition; and a C-terminal cytotoxic domain responsible for channel formation in the cytoplasmic membrane. One domain regulates the target and binds to the receptor on the sensitive cell. The second is involved with translocation, co-opting the machinery of the target cell. The third is the 'killing' domain and may produce a pore in the target cell membrane, or act as a nuclease to chop up the DNA or RNA of the target cell.
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In our system, we design to utilize lysin to realize autolyse of engineered bacteria. It has fur box in promoter and is under regulate of ferrous iron in cytoplasm. After cells autolyse, accumulated DspB and cecropin AD can be released and then kill <i>iron bacteria</i>.
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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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<h1 class="box-heading">Result</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>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>The plasmid was transformed into <i>E. coli</i> BL21, cultured at 37 °C for 12 h, and the plasmid was extracted and verified by PCR.</p>
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<p>To test the expression of DSPB, we cultured E. coli in LB medium containing 0.1% kan. E. coli was cultured at 37℃ and 220 rpm for all night, then inoculated into fresh medium and cultured until logarithmic phase.Add IPTG to final concentration of 20uM and culture E. coli over night at 25℃. Final OD600=0.437(diluted 16 fold) </p>
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<p>In order to test the autolysis effect of Lysin, we constructed the vector pET28a-Lysin. </p>
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<p>The expression of DSPB was verified by SDS-PAGE(Figure). The cell culture medium was blank control, and the pre-induction cell supernatant was used as a negative control. </p>
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<p>From figure 11 we can see that there are bands at about 37 kDa, these band of  experimental groups are thicker than the negative control. And our target protein is about 40kDa. We think the protein here may be DSPB. </p>  
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<p>Then, we tested whether the engineered bacteria expressed active DSPB through enzyme activity experiments. </p>
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<img src="https://static.igem.org/mediawiki/2018/2/22/T--ECUST--result--lysin1.jpg" class="z1">
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<p>4-nitrophenyl-N-acetyl -β-D-glucosaminide(NP-GlcNAc) is hydrolysed by DspB and become 4-nitrophenol with the maximum light absorption at 405 nm. 20 mLbacteria solution is disrupted and acted supernatant as DSPB enzyme solution.
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<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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We washed and shred the bacterial solution after induction, and took the supernatant as the enzyme reaction solution of DSPB while NP-GlcNAc working as a substrate.The enzyme activity experiment was carried out by using 100 uL of enzyme reaction solution and 100 uL of substrate solution (substrate concentration is 5 mM). </p>
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<p>The enzyme activity of the supernatant calculated by the enzyme activity calculation formulais (Activity/(U/mL)=(kOD405-kseldecomposition)*18231.26 *dilution ratio) is 66.363U/mL. </p>
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<p>The plasmid was transformed to <i>E. coli</i> BL21 and cultured at 37 °C for 12h. positive monoclonal bacteria were cultured and verified by PCR. </p>
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<p>The experiment proved that the recombinant bacteria expressed active DSPB, and we used the E. coli supernatant with DSPB activity to carry out the biofilm removal experiment.DH5a was cultured overnight in LB at 37 ° C and 220 rpm, and transferred to a 96-well plate at 37 ° C and cultured for 48 h, then discarded the supernatant .The biofilm was washed with PBS, and 200 uL of the reaction solution was added to react for the whole night. Add crystal violet to stain, wash the solution into the new well plate after alcohol washing, and measure the absorbance at 570 nm. The smaller the absorbance value, the better the membrane removal effect. </p>
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<p>From Fig.13, The supernatant of the recombinant E. coli  has the highest biofilm removal rate, and the biofilm removal effect is getting better with time. </p>
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<img src="https://static.igem.org/mediawiki/2018/2/28/T--ECUST--result--lysin2.jpg" class="z2">
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<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>In order to verify whether the cell expression of Lysin can perforate on the plasma membrane, we test the growth curve of recombinant <i>E.coli</i> and check them under the scanning electron microscope. </p>
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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>
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<img src="https://static.igem.org/mediawiki/2018/c/ca/T--ECUST--result--lysin3.jpg" class="z3">
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<figcaption><b>Figure 3. Cell growth curve with or without IPTG induction. <i>E. coli</i> without IPTG induction grew normally while the density of <i>E.coli</i> decreased after addition of IPTG.</b></figcaption>
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<img src="https://static.igem.org/mediawiki/2018/b/b4/T--ECUST--result--lysin4.jpg" class="z4">
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<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>
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Latest revision as of 02:54, 18 October 2018

Lysin

Description and Design

Lysis gene codes for the colicin-producing bacteria and then activates the expression of colicin. Colicin is a type of bacteriocin produced by and toxic to some strains of Escherichia coli. Colicins are released into the environment to reduce competition from other bacterial strains. Colicins bind to outer membrane receptors, using them to translocate to the cytoplasm or cytoplasmic membrane, where they exert their cytotoxic effect, including depolarisation of the cytoplasmic membrane, DNase activity, RNase activity, or inhibition of murein synthesis. colicins contain at least three domains: an N-terminal translocation domain responsible for movement across the outer membrane and periplasmic space; a central domain responsible for receptor recognition; and a C-terminal cytotoxic domain responsible for channel formation in the cytoplasmic membrane. One domain regulates the target and binds to the receptor on the sensitive cell. The second is involved with translocation, co-opting the machinery of the target cell. The third is the 'killing' domain and may produce a pore in the target cell membrane, or act as a nuclease to chop up the DNA or RNA of the target cell. In our system, we design to utilize lysin to realize autolyse of engineered bacteria. It has fur box in promoter and is under regulate of ferrous iron in cytoplasm. After cells autolyse, accumulated DspB and cecropin AD can be released and then kill iron bacteria.

Result

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.

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.