Composite

Part:BBa_K4621150

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


It consists of the chi18A hypothetical promoter and EctOperon

Usage and Biology

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

chi18A hypothetical promoter is a whole functional sequence including the promoter of chitinase Chi18A 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 Chi18A was up-regulated when SCUT-3 was in chitin medium, and we speculated that its promoter chi18A hypothetical promoter may have properties that can be induced by chitin.

Testing and validation

We ligated the chi18A 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)


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 3369
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 3369
    Illegal NotI site found at 713
    Illegal NotI site found at 1598
    Illegal NotI site found at 1916
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 123
    Illegal XhoI site found at 895
    Illegal XhoI site found at 1069
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 3369
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 3369
    Illegal NgoMIV site found at 930
    Illegal NgoMIV site found at 1336
    Illegal NgoMIV site found at 1342
    Illegal NgoMIV site found at 1633
    Illegal NgoMIV site found at 2682
    Illegal NgoMIV site found at 2874
    Illegal NgoMIV site found at 3384
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 518
    Illegal BsaI.rc site found at 698
    Illegal BsaI.rc site found at 1480
    Illegal BsaI.rc site found at 2008
    Illegal BsaI.rc site found at 2263
    Illegal BsaI.rc site found at 2353
    Illegal BsaI.rc site found at 2428
    Illegal SapI site found at 556


[edit]
Categories
Parameters
None