Difference between revisions of "Team:Shanghai HS ID/Proof Of Concept"

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        <div class="sub-title">Proof of Concept </div>
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        <div class="article-content">
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            Our team plan to use CRISPR-Cas9 to knock out gene LSEI-2094 of Lactobacillus casei ATCC 334, through which
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            we hope to enhance the transformation rate of exogenous DNA in the modified L. casei.
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        </div>
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        <div class="article-content">
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            Here is our designed product development process:<br />
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            · Products of new genetically modified technology<br />
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            · Use CRISPR to target the deletion of some genes.<br />
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            · Cultivation of genetically modified Lactobacillus<br />
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            · Sell technology to companies, companies use technology to produce products.<br />
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            · Product concept: we use CRISPR technology to cultivate new tool bacteria(transgenic Lactobacillus). We
 +
            license the technology to the enterprise. The enterprise uses it, the cost will be reduced.
 +
        </div>
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        <div class="article-content"><b>Supporting Experiment Results</b></div>
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        <div class="article-title">1. Electrophoresis result after PCR</div>
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        <div class="img-wrap no-margin">
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            <img src="https://static.igem.org/mediawiki/2021/4/4c/T--Shanghai_HS_ID--Proof_of_Concept01.jpg" alt="" />
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            <span>Figure 4. Electrophoretogram of pLCNICK PCR resiult</span>
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        </div>
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        <div class="article-content">
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            1-4 are pLCNICK after enzyme digestion, 5 is pLCNICK plasmid before enzyme digestion, 6~7 are upstream
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            homologous arms after PCR, 8~9 are downstream homologous arms after PCR.
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        </div>
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        <div class="article-title">2. Verification result of colony PCR</div>
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        <div class="img-wrap no-margin">
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            <img src="https://static.igem.org/mediawiki/2021/4/47/T--Shanghai_HS_ID--Proof_of_Concept02.jpg" alt="" />
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            <span>Figure 5. PCR verification result</span>
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        </div>
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        <div class="article-content">As showing above, there are bands (1~3, 6, 8~12) at 1000 bp around which are
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            consistent with the DNA profile of the downstream homologous arms. Therefore, it indicated that we have
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            successfully transformed the plasmid in E. coli.</div>
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        <div class="article-title">3. Transformation test result</div>
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        <div class="img-wrap no-margin">
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            <img src="https://static.igem.org/mediawiki/2021/5/5c/T--Shanghai_HS_ID--Proof_of_Concept03.png" alt="" />
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            <span> Graph 1. Comparison between the wild L. caseil and modified L. casei in transformation</span>
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        </div>
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        <div class="article-content">After we electrotransformed the plasmid pIB165 as the foreign DNA to test the
 +
            transformation efficiency of our modified L. casei, it took several days to culture and finally saw the
 +
            comparison results. As showing above, we can see that the modified L. casei (KO) has higher transformation
 +
            efficiency with remarkably more colonies than the wild (Wild).</div>
 +
        <div class="article-content">Clearly it proved that our modified L. casei has a higher transformation rate of
 +
            exogenous DNA. Thus, the experiment results support our product development plan. In the future, we must
 +
            make a series of rigorously designed experiment to further enhance the reliability of our modified L. casei.
 +
            Afterwards, we may then promote the development technique to the market,</div>
 +
        <div class="img-wrap no-margin">
 +
            <img src="https://static.igem.org/mediawiki/2021/3/34/T--Shanghai_HS_ID--Proof_of_Concept04.png" alt="" />
 +
            <span>FFigure 6. Histogram comparison between wild strain and KO strain</span>
 +
        </div>
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        <div class="article-content">
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            In addition, we measured OD<sub>600</sub> of these strain groups which were pre-spreaded plates with
 +
            different volumes
 +
            of bacteria solutions so as to quantify the transformation results as showing above (Fig. 6).<br />
 +
            In conclusion, it indicates that our modified L. casei has much higher efficiency of the foreign plasmid
 +
            transformation than the wild and the modified L. casei has great potential to be used as the recombinant
 +
            carrier in various areas.
  
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Revision as of 18:47, 17 October 2021

Shanghai_HS_ID

Proof of Concept
Our team plan to use CRISPR-Cas9 to knock out gene LSEI-2094 of Lactobacillus casei ATCC 334, through which we hope to enhance the transformation rate of exogenous DNA in the modified L. casei.
Here is our designed product development process:
· Products of new genetically modified technology
· Use CRISPR to target the deletion of some genes.
· Cultivation of genetically modified Lactobacillus
· Sell technology to companies, companies use technology to produce products.
· Product concept: we use CRISPR technology to cultivate new tool bacteria(transgenic Lactobacillus). We license the technology to the enterprise. The enterprise uses it, the cost will be reduced.
Supporting Experiment Results
1. Electrophoresis result after PCR
Figure 4. Electrophoretogram of pLCNICK PCR resiult
1-4 are pLCNICK after enzyme digestion, 5 is pLCNICK plasmid before enzyme digestion, 6~7 are upstream homologous arms after PCR, 8~9 are downstream homologous arms after PCR.
2. Verification result of colony PCR
Figure 5. PCR verification result
As showing above, there are bands (1~3, 6, 8~12) at 1000 bp around which are consistent with the DNA profile of the downstream homologous arms. Therefore, it indicated that we have successfully transformed the plasmid in E. coli.
3. Transformation test result
Graph 1. Comparison between the wild L. caseil and modified L. casei in transformation
After we electrotransformed the plasmid pIB165 as the foreign DNA to test the transformation efficiency of our modified L. casei, it took several days to culture and finally saw the comparison results. As showing above, we can see that the modified L. casei (KO) has higher transformation efficiency with remarkably more colonies than the wild (Wild).
Clearly it proved that our modified L. casei has a higher transformation rate of exogenous DNA. Thus, the experiment results support our product development plan. In the future, we must make a series of rigorously designed experiment to further enhance the reliability of our modified L. casei. Afterwards, we may then promote the development technique to the market,
FFigure 6. Histogram comparison between wild strain and KO strain
In addition, we measured OD600 of these strain groups which were pre-spreaded plates with different volumes of bacteria solutions so as to quantify the transformation results as showing above (Fig. 6).
In conclusion, it indicates that our modified L. casei has much higher efficiency of the foreign plasmid transformation than the wild and the modified L. casei has great potential to be used as the recombinant carrier in various areas.
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