Difference between revisions of "Team:ECUST/Biocide"

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<div id="bannerquote">BIOCIDE</div>
 
<div id="bannerquote">BIOCIDE</div>
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<h1 class="box-heading">Description</h1>
 
<h1 class="box-heading">Description</h1>
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<p>When the concentration of Fe3+ in the environment is high, it means that the hard skeleton of the microenvironment of the iron bacteria is destroyed and loses protection. At this time, the added bactericide will have a greater killing effect on the bacteria. The bactericide we selected was cecropin AD, a cationic chimeric antimicrobial peptide obtained from cecropin, consisting of the first 11 amino acid residues of cecropin A and the last 26 amino acid residues of cecropin D. It has strong antibacterial activity against Gram-positive and negative bacteria.</p>
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<h1 class="box-heading">Result</h1>
 
<p>We insert fragment of Cecropin AD into vector pET-28a(Figure 1) </p>
 
<p>We insert fragment of Cecropin AD into vector pET-28a(Figure 1) </p>
 
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       <img src="https://static.igem.org/mediawiki/2018/a/a5/T--ECUST--Biocide1.jpg" class="zoom">
 
       <img src="https://static.igem.org/mediawiki/2018/a/a5/T--ECUST--Biocide1.jpg" class="zoom">
 
       <figcaption><b>Figure 1. The vector pET-28a Vector is cut by NcoI and BamHI. Sequence of AD was chemically synthesized and amplified by PCR, then 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 AD was chemically synthesized and amplified by PCR, then 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 12 h. positive monoclonal bacteria were cultured and verified by PCR(Figure 2). </p>
 
<p>The plasmid was transformed to E. coli BL21 and cultured at 37 °C for 12 h. positive monoclonal bacteria were cultured and verified by PCR(Figure 2). </p>
 
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       <img src="https://static.igem.org/mediawiki/2018/c/c6/T--ECUST--Biocide2.jpg" class="zoom">
 
       <img src="https://static.igem.org/mediawiki/2018/c/c6/T--ECUST--Biocide2.jpg" class="zoom">
 
       <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>We verified the expression of Cecropin AD by SDS-PAGE(Figure 3). </p>
 
<p>We verified the expression of Cecropin AD by SDS-PAGE(Figure 3). </p>
 
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       <img src="https://static.igem.org/mediawiki/2018/8/86/T--ECUST--Biocide3.jpg" class="zoom">
 
       <img src="https://static.igem.org/mediawiki/2018/8/86/T--ECUST--Biocide3.jpg" class="zoom">
 
       <figcaption><b>Figure 3. The SDS-PAGE of Cecropin AD
 
       <figcaption><b>Figure 3. The SDS-PAGE of Cecropin AD
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<p>To test the effect of cecropin AD, we measured the growth curve of iron bacteria adding with different concentration of cecropin AD.</p>
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<p>We can see target band in SDS-PAGE. However, when we break engineered bacteria and use broken-bacteria supernatant to characterize, we find that it does not have the function of killing bacteria. So we synthesize cecropin AD from Genscript. Then antibacterial experiment is performed.</p>
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<p>Iron bacteria grows adding with different concentration of cecropin AD, and growth curve is measured at the light absorption of 600nm.</p>
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<p>From the figure, we can see that cecropin AD can repress growth of bacteria to a certain extent.</p>
 
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       <img src="https://static.igem.org/mediawiki/2018/3/30/T--ECUST--Biocide4.jpg" class="zoom">
 
       <img src="https://static.igem.org/mediawiki/2018/3/30/T--ECUST--Biocide4.jpg" class="zoom">
 
       <figcaption><b>Figure 4. Growth curve of iron bacteria adding with different concentration of cecropin AD</b></figcaption>         
 
       <figcaption><b>Figure 4. Growth curve of iron bacteria adding with different concentration of cecropin AD</b></figcaption>         

Latest revision as of 03:48, 18 October 2018

BIOCIDE

Description

When the concentration of Fe3+ in the environment is high, it means that the hard skeleton of the microenvironment of the iron bacteria is destroyed and loses protection. At this time, the added bactericide will have a greater killing effect on the bacteria. The bactericide we selected was cecropin AD, a cationic chimeric antimicrobial peptide obtained from cecropin, consisting of the first 11 amino acid residues of cecropin A and the last 26 amino acid residues of cecropin D. It has strong antibacterial activity against Gram-positive and negative bacteria.

Result

We insert fragment of Cecropin AD into vector pET-28a(Figure 1)

Figure 1. The vector pET-28a Vector is cut by NcoI and BamHI. Sequence of AD was chemically synthesized and amplified by PCR, then ligated with linearized vector by Ezmax.

The plasmid was transformed to E. coli BL21 and cultured at 37 °C for 12 h. positive monoclonal bacteria were cultured and verified by PCR(Figure 2).

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

We verified the expression of Cecropin AD by SDS-PAGE(Figure 3).

Figure 3. The SDS-PAGE of Cecropin AD Lane 1 :before induction Lane 2,3:after induction

We can see target band in SDS-PAGE. However, when we break engineered bacteria and use broken-bacteria supernatant to characterize, we find that it does not have the function of killing bacteria. So we synthesize cecropin AD from Genscript. Then antibacterial experiment is performed.

Iron bacteria grows adding with different concentration of cecropin AD, and growth curve is measured at the light absorption of 600nm.

From the figure, we can see that cecropin AD can repress growth of bacteria to a certain extent.

Figure 4. Growth curve of iron bacteria adding with different concentration of cecropin AD

The figure shows that cecropin AD has powerful bacterividal activity. Only concentration of cecropin AD is low enough, can iron bacteria grow.