Difference between revisions of "Team:XJTU-China/Improve"

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<h1>Improve</h1>
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<p>For teams seeking to improve upon a previous part or project, you should document all of your work on this page. Please remember to include all part measurement and characterization data on the part page on the Registry. Please include a link to your improved part on this page.</p>
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<h3>Gold Medal Criterion #2</h3>
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<p><b>Standard Tracks:</b> Create a new part that has a functional improvement upon an existing BioBrick part. The sequences of the new and existing parts must be different. You must perform experiments with both parts to demonstrate this improvement. Document the experimental characterization on the Part's Main Page on the Registry for both the existing and new parts. Both the new and existing Main Page of each Part’s Registry entry must reference each other. Submit a sample of the new part to the Registry.
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The existing part must NOT be from your 2018 part number range and must be different from the part documented in bronze #4.
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<b>Special Tracks:</b> Improve the function of an existing iGEM project (that your current team did not originally create) and display your achievement on your wiki.</p>
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        <h1 class="font-weight-bold text-center">Improvement</h1>
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        <p>
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          1. mcherry reporter is changed into EGFP as the consideration of the strength of EGFP is stronger than mcherry, meanwhile EGFP is more sensitive and detectable than mcherry by microplate reader in realistic conditions. Figure 1 shows intensity of mcherry in previous BBa_K2448025(link), and we know that it is rather hard to see strength changes in the first few concentration gradients and the spike around 1mM psicose is really hard to illustrate.
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        <img class="mx-auto d-block" src="https://static.igem.org/mediawiki/2017/d/d9/T--Evry_Paris-Saclay--pPsiA-PsiR-At.png" />
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          Figure 1.Changes of fluorescence intensity per OD with different concentration of psicose (Data cited from <a href="http://parts.igem.org/Part:BBa_K2448025">BBa_K2448025</a>)
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          Compared with mcherry, EGFP fluorescence intensity is 100 times stronger than mcherry. And we could distinguish differences of fluorescence per ABS600 within 5mM psicose easily, as shown in  Figure 2. As a results, the detection range of our sensing circuit is wider than previous parts and our part is more sensitive.
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        <img class="mx-auto d-block" src="https://static.igem.org/mediawiki/2018/1/12/T--XJTU-China--Demo-2.png" />
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        <p class="text-center">
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          Figure 2 Dose effect of D-psicose' s effect on pPsi promoter (Our improved data)
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        <p>
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          2.  We have made some improvements in the experiment design. In addition to testify the effect of fructose on pPsi promoter in in previous BBa_K2448025 (Figure 3), we measured effect of fructose and IPTG, which make our design more complete and persuasive (Figure 4). Figure 4 shows that at the same timepoint, the fluorescence intensity varies with different sugar, and psicose is always higher than other sugar, while IPTG has a little effect to induce pPsi promoter. So we can draw the conclusion that pPsi promoter is specific to D-psicose and is a little sensitive to IPTG in this system.
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        <img class="mx-auto d-block" src="https://static.igem.org/mediawiki/2017/e/e3/T--Evry_Paris-Saclay--pPsiA-PsiR-At_Fru-Psi.png" />
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        <p class="text-center">
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          Figure 3 Influence of fructose in previous part (Data cited from <a href="http://parts.igem.org/Part:BBa_K2448025">BBa_K2448025</a>)
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        <img class="mx-auto d-block" src="https://static.igem.org/mediawiki/2018/9/95/T--XJTU-China--Demo-1.png" />
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        <p class="text-center">
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          Figure 4 Effect of different sugars on pPsi promoter (Our improved data)
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        </p>
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        <p>
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          3.  We changed the direction of reporter gene, as shown in Fig 5. In previous BBa_K2448025, The operons of PsiR (promoted by a constative promoter) and EGFP (promoted by pPsi) are in the same direction, but in our circuit they are in the reverse direction, in order to minimize read-through of terminators and to prevent homologous recombination of the plasmid itself. As this circuit will be present in chassis cells under high growth pressure, the cell or plasmid could undergo unexpected mutations to maximize its survival. This design is meant to prevent such things from happening.
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        </p>
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        <img class="mx-auto d-block" src="https://static.igem.org/mediawiki/2018/3/31/T--XJTU-China--Project-device-a.jpg" />
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        <p class="text-center">
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          Fig 5 Design of our sensing circuit (<a href="http://parts.igem.org/Part:BBa_K2791004">BBa_K2791004</a>)
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Latest revision as of 03:37, 18 October 2018

Improvement

1. mcherry reporter is changed into EGFP as the consideration of the strength of EGFP is stronger than mcherry, meanwhile EGFP is more sensitive and detectable than mcherry by microplate reader in realistic conditions. Figure 1 shows intensity of mcherry in previous BBa_K2448025(link), and we know that it is rather hard to see strength changes in the first few concentration gradients and the spike around 1mM psicose is really hard to illustrate.

Figure 1.Changes of fluorescence intensity per OD with different concentration of psicose (Data cited from BBa_K2448025)

Compared with mcherry, EGFP fluorescence intensity is 100 times stronger than mcherry. And we could distinguish differences of fluorescence per ABS600 within 5mM psicose easily, as shown in Figure 2. As a results, the detection range of our sensing circuit is wider than previous parts and our part is more sensitive.

Figure 2 Dose effect of D-psicose' s effect on pPsi promoter (Our improved data)

2. We have made some improvements in the experiment design. In addition to testify the effect of fructose on pPsi promoter in in previous BBa_K2448025 (Figure 3), we measured effect of fructose and IPTG, which make our design more complete and persuasive (Figure 4). Figure 4 shows that at the same timepoint, the fluorescence intensity varies with different sugar, and psicose is always higher than other sugar, while IPTG has a little effect to induce pPsi promoter. So we can draw the conclusion that pPsi promoter is specific to D-psicose and is a little sensitive to IPTG in this system.

Figure 3 Influence of fructose in previous part (Data cited from BBa_K2448025)

Figure 4 Effect of different sugars on pPsi promoter (Our improved data)

3. We changed the direction of reporter gene, as shown in Fig 5. In previous BBa_K2448025, The operons of PsiR (promoted by a constative promoter) and EGFP (promoted by pPsi) are in the same direction, but in our circuit they are in the reverse direction, in order to minimize read-through of terminators and to prevent homologous recombination of the plasmid itself. As this circuit will be present in chassis cells under high growth pressure, the cell or plasmid could undergo unexpected mutations to maximize its survival. This design is meant to prevent such things from happening.

Fig 5 Design of our sensing circuit (BBa_K2791004)

Copyright © 2018 XJTU-China iGEM Team. All rights reserved.