Composite

Part:BBa_K4621152

Designed by: Anyang Chen   Group: iGEM23_SCUT-China   (2023-10-05)
Revision as of 18:04, 10 October 2023 by Arml (Talk | contribs) (→‎Supplementary work in this module)


It consists of the chi19C hypothetical promoter and EctOperon.

Usage, Biology and Characterization

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

chi19C hypothetical promoter is a functional fragment in chitinase Chi19C 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 Chi19C was up-regulated when SCUT-3 was in chitin medium, and we speculated that its promoter chi19C hypothetical promoter may have properties that can be induced by chitin.

Testing and validation

We ligated the chi19C 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. In our later shrimp shell fermentation, it again proved our decision here to be reasonable and crucial, which would be shown later.

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.

Supplementary work in this module

After confirmation of their inducibility, we further conducted experiments to test if the induction of ectione operon could be achieved at different time points in a relative long-term fermentation, considering that the shrimp shell recycling fermentation typically last five-day long.

effect-of-different-induction-times-on-yield-c.png

Fig.2 Fermentation of the product of SCUT-3-EctC at different induction times


The fermentation experiments in this section were all conducted on the basis of using LB media, and there was no chitin added in the Ctrl group. In the fellow groups, 1% chitin powder was added at different time points, namely 0 d, 1 d, 2 d, 3 d, and 4 d. The test results indicated that the induction of ProC could be achieved in a comparatively early stage from day 0 to day 2.



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 3468
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 3468
    Illegal NotI site found at 812
    Illegal NotI site found at 1697
    Illegal NotI site found at 2015
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 93
    Illegal XhoI site found at 994
    Illegal XhoI site found at 1168
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3468
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3468
    Illegal NgoMIV site found at 1029
    Illegal NgoMIV site found at 1435
    Illegal NgoMIV site found at 1441
    Illegal NgoMIV site found at 1732
    Illegal NgoMIV site found at 2781
    Illegal NgoMIV site found at 2973
    Illegal NgoMIV site found at 3483
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 617
    Illegal BsaI.rc site found at 797
    Illegal BsaI.rc site found at 1579
    Illegal BsaI.rc site found at 2107
    Illegal BsaI.rc site found at 2362
    Illegal BsaI.rc site found at 2452
    Illegal BsaI.rc site found at 2527
    Illegal SapI site found at 655


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