Difference between revisions of "Team:H14Z1 Hangzhou/Demonstrate"

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<h1>Demonstrate</h1>
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Compared with the standard spectrogram, the peak time of SAM product is about 6.0, and the internal standard is added to confirm the peak shape of SAM. Because SAM exists widely in all kinds of organisms, there is also a SAM peak in contrast. However, the SAM yield of engineered bacteria was two times that of the control. Unfortunately, the SAM2 gene from yeast has not been able to detect the increase in yield. It was found that SAM2 gene contained many rare codons of lactic acid bacteria, which affected translation and resulted in no protein expression.
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<h3>Gold Medal Criterion #4</h3>
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<p>
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your project works. There are many ways in which your project working could be demonstrated, so there is more than one way to meet this requirement. This gold medal criterion was introduced in 2016, so check our what 2016 teams did to achieve their gold medals!
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Please see the <a href="https://2018.igem.org/Judging/Medals">2018 Medals Page</a> for more information.
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            <img src="https://static.igem.org/mediawiki/2018/8/86/T--H14Z1_Hangzhou--head_demonstrate.png" alt="" class="head_div_img" />
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            <div class="content_box">
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                <h1 class="content_title">Demonstrate</h1>
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                <div class="content_conts">
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                    <!------------------------HPLC sample------------------------------ -->
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                    <h3 class="content_subtitle">HPLC sample</h3>
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                    <p class="mt8 content_context">
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                        <span style="width:40px">Fig1.</span><span style="width:500px">GSH standard products peak at
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                            7.5min</span>
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                    </p>
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                    <p class="mt8 content_context">
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                        <span style="width:40px">Fig2.</span><span style="width:500px">NZ9000 intracellular extracts
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                            from no-load cells</span>
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                    </p>
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                    <p class="mt8 content_context">
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                        <span style="width:40px">Fig3.</span><span style="width:500px">intracellular extracts
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                            of.NZ9000/pNZ-gshF</span>
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                    </p>
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                    <p><img style="width: 60%;" src="https://static.igem.org/mediawiki/2018/4/44/T--H14Z1_Hangzhou--results_demonstrate_fig1.PNG"></p>
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                    <p class="content_context" style="text-indent:2em; text-align:justify;">
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                        According to the area calculation, the product was determined by internal standard
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                        method. Sample size and standard volume are mixed for testing.
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                        The results show that the sealing area is not much different at about 7.45 min, but the area of
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                        7.75 min is half of the original. It is verified that the peak is the product peak of GSH.
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                    </p>
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                    <p><img style="width: 60%;" src="https://static.igem.org/mediawiki/2018/c/c1/T--H14Z1_Hangzhou--results_demonstrate_fig2.PNG"></p>
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                    <p class="mt8 content_context" style="text-align:center">
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                        <span style="width:80px">Fig4.</span><span>GSH concentration / cell dry
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                            weight</span>
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                    </p>
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                    <p class="content_context" style="text-indent:2em; text-align:justify;">
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                        Comparing the GSH yields of the two groups, the first group was lower than the second group in
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                        general, and both groups had the highest GSH yields after 6 hours of induction. Up to 8.62mg/g.
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                    </p>
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                    <!------------------------HPLC detection of SAM------------------------------ -->
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                    <h3 class="content_subtitle">HPLC detection of SAM</h3>
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                    <p class="content_context" style="text-indent:2em; text-align:justify;">
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                        Fermentation conditions: when OD600=0.4 was added with different concentration of inducer, the
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                        final concentration was 20ng/ml, 50ng/ml, 100ng/ml nisin was induced and methionine at the
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                        final concentration of 1g/l was added
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                        as substrate.
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                    </p>
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                    <p>
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                        <span class="content_context">a：20ng/ml</span>
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                        <span class="content_context">b：50ng/ml</span>
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                        <span class="content_context">c：100ng/ml</span>
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                    </p>
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                    <p><img style="width: 60%;" src="https://static.igem.org/mediawiki/2018/e/ed/T--H14Z1_Hangzhou--results_demonstrate_fig3.PNG"></p>
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                    <p class="mt8 content_context" style="text-align:center">
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                        <span style="width:80px">Fig5.</span><span>SAM liquid phase detection chart</span>
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                    </p>
  
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                    <p><img style="width: 60%;" src="https://static.igem.org/mediawiki/2018/8/89/T--H14Z1_Hangzhou--results_demonstrate_fig4.PNG"></p>
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                    <p class="mt8 content_context" style="text-align:center">
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                        <span style="width:80px">Fig6.</span><span>SAM2 production in
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                            NZ9000/pNZ-metk cells</span>
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                    </p>
  
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                    <p class="content_context" style="text-indent:2em; text-align:justify;">
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                        Fig6. shows that although there is no significant metK protein on SDS-PAGE, the intracellular
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                        SAM production is increased in varying degrees at different induction concentrations. The
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                        highest yield of 50 ng/ml is 15.2 mg/g.
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Revision as of 23:07, 13 October 2018


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Demonstrate

HPLC sample

Fig1.GSH standard products peak at 7.5min

Fig2.NZ9000 intracellular extracts from no-load cells

Fig3.intracellular extracts of.NZ9000/pNZ-gshF

According to the area calculation, the product was determined by internal standard method. Sample size and standard volume are mixed for testing. The results show that the sealing area is not much different at about 7.45 min, but the area of 7.75 min is half of the original. It is verified that the peak is the product peak of GSH.

Fig4.GSH concentration / cell dry weight

Comparing the GSH yields of the two groups, the first group was lower than the second group in general, and both groups had the highest GSH yields after 6 hours of induction. Up to 8.62mg/g.

HPLC detection of SAM

Fermentation conditions: when OD600=0.4 was added with different concentration of inducer, the final concentration was 20ng/ml, 50ng/ml, 100ng/ml nisin was induced and methionine at the final concentration of 1g/l was added as substrate.

a：20ng/ml b：50ng/ml c：100ng/ml

Fig5.SAM liquid phase detection chart

Fig6.SAM2 production in NZ9000/pNZ-metk cells

Fig6. shows that although there is no significant metK protein on SDS-PAGE, the intracellular SAM production is increased in varying degrees at different induction concentrations. The highest yield of 50 ng/ml is 15.2 mg/g.