Composite

Part:BBa_K4621132

Designed by: Anyang Chen, Ruikang Chen   Group: iGEM23_SCUT-China   (2023-10-05)
Revision as of 03:52, 6 October 2023 by Registry (Talk | contribs)

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Complex functional parts of SCUT-3-EctPi8 that induce high Hydroxyectoine production.

Usage, Biology and Characterization

LPMO hypothetical promoter is a whole functional sequence in front of the Lytic polysaccharide monooxygenase LPMO 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 monooxygenase LPMO was up-regulated when SCUT-3 was in chitin medium, and we speculated that its promoter Pro lpmo may have properties that can be induced by chitin.

This CRISPRi fragment contains four parts : BBa _ K4621070, BBa _ K3875019, BBa _ K4621031, and BBa _ K3081104, covering the dCas9 and sgRNA necessary for the CRISPRi system. The CRISPRi system is suitable for a variety of Streptomyces.[1] In this study, it was used to inhibit the expression of GQS52 _ 08040 gene in SCUT-3 to produce more ectoine and hydroxyectoine. mechanism-of-crispri.png

Fig.1Mechanism of CRISPRi

Testing and validation

Due to the limited types of plasmids available for SCUT-3, we inserted the CRISPRi fragment into the previously constructed plasmid PZ-ProP-Ect-i8 by homologous recombination to achieve simultaneous stable expression of multiple target genes. Subsequently, we verified the effectiveness of the CRISPRi system in the fermentation of ordinary LB, high-salt LB and shrimp shells. fermentation-under-high-salt-environment.png

Fig.2 Fermentation under the high salt environment

fermentation-using-shrimp-shells-p.png

Fig.3 Fermentation using shrimp shells

Reference

[1] Zhao Y, Li L, Zheng G, Jiang W, Deng Z, Wang Z, Lu Y. CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces. Biotechnol J. 2018 Sep;13(9):e1800121. doi: 10.1002/biot.201800121. Epub 2018 Jul 4. PMID: 29862648.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 3756
    Illegal SpeI site found at 8275
    Illegal PstI site found at 3437
    Illegal PstI site found at 3744
    Illegal PstI site found at 6169
    Illegal PstI site found at 6403
    Illegal PstI site found at 7615
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 8252
    Illegal SpeI site found at 8275
    Illegal PstI site found at 3437
    Illegal PstI site found at 3744
    Illegal PstI site found at 6169
    Illegal PstI site found at 6403
    Illegal PstI site found at 7615
    Illegal NotI site found at 781
    Illegal NotI site found at 1666
    Illegal NotI site found at 1984
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4208
    Illegal BglII site found at 5003
    Illegal BglII site found at 7772
    Illegal BamHI site found at 3948
    Illegal BamHI site found at 5297
    Illegal BamHI site found at 8240
    Illegal BamHI site found at 8445
    Illegal XhoI site found at 963
    Illegal XhoI site found at 1137
    Illegal XhoI site found at 4621
    Illegal XhoI site found at 6967
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 3756
    Illegal SpeI site found at 8275
    Illegal PstI site found at 3437
    Illegal PstI site found at 3744
    Illegal PstI site found at 6169
    Illegal PstI site found at 6403
    Illegal PstI site found at 7615
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 3756
    Illegal SpeI site found at 8275
    Illegal PstI site found at 3437
    Illegal PstI site found at 3744
    Illegal PstI site found at 6169
    Illegal PstI site found at 6403
    Illegal PstI site found at 7615
    Illegal NgoMIV site found at 998
    Illegal NgoMIV site found at 1404
    Illegal NgoMIV site found at 1410
    Illegal NgoMIV site found at 1701
    Illegal NgoMIV site found at 2750
    Illegal NgoMIV site found at 2942
    Illegal NgoMIV site found at 3452
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 586
    Illegal BsaI.rc site found at 766
    Illegal BsaI.rc site found at 1548
    Illegal BsaI.rc site found at 2076
    Illegal BsaI.rc site found at 2331
    Illegal BsaI.rc site found at 2421
    Illegal BsaI.rc site found at 2496
    Illegal SapI site found at 624
    Illegal SapI site found at 4539
    Illegal SapI site found at 5829
    Illegal SapI.rc site found at 7026


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Categories
Parameters
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