Composite

Part:BBa_K4621152

Designed by: Anyang Chen   Group: iGEM23_SCUT-China   (2023-10-05)
Revision as of 17:59, 5 October 2023 by Arml (Talk | contribs)


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 120h. 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(72h)

By adding chitin at different days after fermentation, we also determined that the optimal time for chitin induction of chi19C hypothetical promoter was the second day of fermentation, and the specific experimental data are shown in FIG. 2: effect-of-different-induction-times-on-yield-c.png

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

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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