Composite

Part:BBa_K4621151

Designed by: Anyang Chen   Group: iGEM23_SCUT-China   (2023-10-05)
Revision as of 18:16, 10 October 2023 by Arml (Talk | contribs)

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It consists of the chi18B hypothetical promoter and EctOperon.

Usage, Biology and Characterization

EctOperon contains ectA, ectB, ectC and ectD and can produce 5-Hydroxyectoine from lysine.

chi18B hypothetical promoter is a functional fragment in chitinase Chi18B gene, which was cloned from SCUT-3 gene. In our project, through transcriptome analysis, qPCR and other means, we found that the gene expression of chitinase Chi18B was up-regulated when SCUT-3 was in chitin medium, and we speculated that its promoter chi18B hypothetical promoter may have properties that can be induced by chitin.

Testing and validation

We ligated the chi18B hypothetical promoter with EctOperon and overexpressed it in Streptomyces SCUT-3 using an integrated plasmid. The modified strain was inoculated in 40 mL LB medium and fermented at 40℃ and 220 rpm for 48h. The production of Ectoine and 5-Hydroxyectoin in the presence or without chitin (the first day of fermentation) was detected to evaluate the chitin inducability of the promoter. The results are shown in FIG. 1. effect-of-different-promoters-on-yield.png

Fig.1 Fermentation of products with or without the addition of an inducer（chitin) (a).Ectoine (b).5-Hydroxyectoin(48h)


The performance test indicated that ectoine production showed almost no significant difference between induced condition and non-induced condition, but hydroxyectoine varies in production level and severity of inducibility. ProP, a nickname for promoter of LPMO, and ProC, a nickname for promoter of chi19C, presented the two most strict inducibility among four promoters tested. Besides, ProP and ProA, a nickname for promoter of chi18A, shows two greatest strength.

From our results, it’s easy to assume that ProP is the most applicable promoter to continue studying. At the same time, we gave the runner-up to ProC. ProA, with great leakage of expression, was insufficient for further engineering, considering that the chemical we aim to produce could be harmful for host cells’ growth, so we put severity over strength when assessing performance of promoters.

The production of ectoine and hydroxyectoine tested in this experiment was relative limited, up to about 60-70mg/L. Further optimization and scale-up attempts could be made to ensure the strains can effectively valorize shrimp shell in industrial processing.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3567
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 3567
    Illegal NotI site found at 911
    Illegal NotI site found at 1796
    Illegal NotI site found at 2114
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1093
    Illegal XhoI site found at 1267
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3567
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3567
    Illegal NgoMIV site found at 1128
    Illegal NgoMIV site found at 1534
    Illegal NgoMIV site found at 1540
    Illegal NgoMIV site found at 1831
    Illegal NgoMIV site found at 2880
    Illegal NgoMIV site found at 3072
    Illegal NgoMIV site found at 3582
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 716
    Illegal BsaI.rc site found at 896
    Illegal BsaI.rc site found at 1678
    Illegal BsaI.rc site found at 2206
    Illegal BsaI.rc site found at 2461
    Illegal BsaI.rc site found at 2551
    Illegal BsaI.rc site found at 2626
    Illegal SapI site found at 754


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