Difference between revisions of "Team:XJTLU-CHINA/Demonstrate"
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<img width="650px" src="https://static.igem.org/mediawiki/2017/4/43/Gel_electrophoresis.jpeg"> | <img width="650px" src="https://static.igem.org/mediawiki/2017/4/43/Gel_electrophoresis.jpeg"> | ||
<figcaption> | <figcaption> | ||
| − | <strong>From left to right: pSB1C3+LL-37 (cut by PstI, uncut); pSB1C3+GF-17 (cut by pstI, uncut); 1kb DNA maker; | + | <strong>Fig.1 From left to right: pSB1C3+LL-37 (cut by PstI, uncut); pSB1C3+GF-17 (cut by pstI, uncut); 1kb DNA maker; |
pEt28a (+)+3x AMPs (uncut, cut)</strong> | pEt28a (+)+3x AMPs (uncut, cut)</strong> | ||
</figcaption> | </figcaption> | ||
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</div> | </div> | ||
<div class="para"> | <div class="para"> | ||
| − | <h2> | + | <h2>2. Expression of LL-37 in bacteria screened by dot blotting</h2> |
</div> | </div> | ||
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<img width="650px" src="https://static.igem.org/mediawiki/2017/7/7a/Demonstration_9.png"> | <img width="650px" src="https://static.igem.org/mediawiki/2017/7/7a/Demonstration_9.png"> | ||
</figure> | </figure> | ||
| + | <figcaption> | ||
| + | <strong>Fig. 2 time gradients for dot bolt. The left shows the protein were produced and right is non-induced (negative control). The result shows that whole circuit can be induced by IPTG | ||
| + | </strong> | ||
| + | </figcaption> | ||
</div> | </div> | ||
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</p> | </p> | ||
| − | <h2> | + | <h2>4. Determination of the minimal inhibitory concentration (MIC) of the three AMPs.</h2> |
</div> | </div> | ||
<div class="fig" align="center"> | <div class="fig" align="center"> | ||
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<div class="para"> | <div class="para"> | ||
| − | <h2> | + | <h2>5. Absorbance (cell density) of the biofilm of |
<i>S. aureus</i> against different concentrations of the three individual AMPs or a mixture of them </h2> | <i>S. aureus</i> against different concentrations of the three individual AMPs or a mixture of them </h2> | ||
</div> | </div> | ||
Revision as of 02:06, 2 November 2017
Demonstration
For the whole circuit construct, we did following experiments:
1. Gel electrophoresis
2. Expression of LL-37 in bacteria screened by dot blotting
3. Inhibition Ring
This experiment qualitatively evaluate the effective of Anti-microbial peptides (AMPs). The result shows that the AMPs has effective on Staphylococcus aureus.
Figure 1 shows the AMPs has effective on Staphylococcus aureus, It seems that all of three AMPs has effective on Staphylococcus aureus.
Aimed at examining the efficacy of the antimicrobial peptides on killing S. aureus (each experiment below were repeated 3 times).
4. Determination of the minimal inhibitory concentration (MIC) of the three AMPs.
Both single peptide of the three AMPs and the mixture of them have conspicuous lethal effect on S. aureus.
5. Absorbance (cell density) of the biofilm of S. aureus against different concentrations of the three individual AMPs or a mixture of them
The biofilm formation of S. aureus was to a great extent inhibited by these three AMPs.
Experience and conclusions:
- Peptides has shown their antimicrobial activity on S. aureus. And they can successfully expressed in E. coli.
- AgrA, which is an essential protein in quorum sensing construct, signifies toxicity to cells during growth.
- We have trouble in obtaining enough plasmid DNA extracted from L. lactis after transformation, probably due to either low copy number or low yield from the extraction protocol.
Collaborators and Supporters
