Difference between revisions of "Part:BBa K4621096"

 
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<partinfo>BBa_K4621096 short</partinfo>
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===Usage, Biology and Characterization===
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Pro1008 contains the promoter（BBa_K3880007） of GQS52_24880 in SCUT-3 without 5’-UTR. The fusion promoter composed of Pro1008 and chitin-inducible promoter can control the gene expression switch while controlling its expression intensity.
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===Testing and validation===
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Pro1008 was inserted into the chitin-inducible promoter ProP to form a fusion promoter. The regulation of gene expression can be achieved by placing it before the coding of the target gene. In our study, Pro1008 stably expressed the target gene under the premise of chitin induction to control the production of ectoine and hydroxyectoine. We verified its regulation effect by LB fermentation.
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For the experimenting group, we added chitin at 24 h to induce expression, and extended the total fermentation time to 5 days, to test the general accumulation.
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https://static.igem.wiki/teams/4621/wiki/parts/fermentation-with-an-inducible-promoter.png
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We are surprising by the effect when combining the ProP with p24880 (p1007 & p1008), however, the results are seemingly incompatible with other data that higher expression leads to decrease on production. This phenomenon may be explained by the hypothesis of severe overexpressing leading to rapid reduction on growth and survival of SCUT-3, that p24880 carrier yield a large amount of ectoine and hydroxyectoine once induced by chitin, leading to immediate reduction in the general metabolism due to loss of living bacteria. If this case is true, p24880 is still not sufficient for long-period fermentation, and should be optimized sorely.
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We also observed that p24880-UTRless (p1008) can still produce ectoine and hydroxyectoine despite the absence of 5’ UTR, from which we reasonably assume that in ectoine operon there are other operator lying in genes of ecB, ectC or thpD that is essential for production, which needs further investigation and genome analysis.

Revision as of 19:28, 10 October 2023


Fusion promoter composed of Pro1008 and ProP

Usage, Biology and Characterization

Pro1008 contains the promoter（BBa_K3880007） of GQS52_24880 in SCUT-3 without 5’-UTR. The fusion promoter composed of Pro1008 and chitin-inducible promoter can control the gene expression switch while controlling its expression intensity.

Testing and validation

Pro1008 was inserted into the chitin-inducible promoter ProP to form a fusion promoter. The regulation of gene expression can be achieved by placing it before the coding of the target gene. In our study, Pro1008 stably expressed the target gene under the premise of chitin induction to control the production of ectoine and hydroxyectoine. We verified its regulation effect by LB fermentation.

For the experimenting group, we added chitin at 24 h to induce expression, and extended the total fermentation time to 5 days, to test the general accumulation. fermentation-with-an-inducible-promoter.png


We are surprising by the effect when combining the ProP with p24880 (p1007 & p1008), however, the results are seemingly incompatible with other data that higher expression leads to decrease on production. This phenomenon may be explained by the hypothesis of severe overexpressing leading to rapid reduction on growth and survival of SCUT-3, that p24880 carrier yield a large amount of ectoine and hydroxyectoine once induced by chitin, leading to immediate reduction in the general metabolism due to loss of living bacteria. If this case is true, p24880 is still not sufficient for long-period fermentation, and should be optimized sorely.

We also observed that p24880-UTRless (p1008) can still produce ectoine and hydroxyectoine despite the absence of 5’ UTR, from which we reasonably assume that in ectoine operon there are other operator lying in genes of ecB, ectC or thpD that is essential for production, which needs further investigation and genome analysis.