Composite

Part:BBa_K4621130

Designed by: Anyang Chen, Ruikang Chen   Group: iGEM23_SCUT-China   (2023-10-05)
Revision as of 11:41, 11 October 2023 by Conerrr (Talk | contribs) (→‎Usage, Biology and Characterization)

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Complex functional parts of SCUT-3-EctPi2 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 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-i2 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


Under 1% NaCl, CRISPRi-introduced strains show decrease on the total production when compared to ProP, and for Pi4 and Pi8, they nearly offset the positive effect of ProP overexpressing. This result did not align with our expectation. To further invest about which factor influence the performance of CRISPRi, we conducted another experience which replace the common LB medium to LB medium with 5% NaCl. We wished to test if the performance was affected by insufficient metabolic flux.

As shown in Figure 2B, under 5% NaCl, CRISPRi indroduced strains Pi4 showed significant increase among all strains tested.


fermentation-using-shrimp-shells-p.png

Fig.3 Fermentation using shrimp shells


From Figure 3A we can see that Pi4 creates the highest yield of ectoine and hydroxyecyoine. In amino acid analysis of Figure 3B, we can see that the shrimp shell degradation level in modified bacteria is even higher than wild type.

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 3741
    Illegal SpeI site found at 8260
    Illegal PstI site found at 3437
    Illegal PstI site found at 3729
    Illegal PstI site found at 6154
    Illegal PstI site found at 6388
    Illegal PstI site found at 7600
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 8237
    Illegal SpeI site found at 8260
    Illegal PstI site found at 3437
    Illegal PstI site found at 3729
    Illegal PstI site found at 6154
    Illegal PstI site found at 6388
    Illegal PstI site found at 7600
    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 4193
    Illegal BglII site found at 4988
    Illegal BglII site found at 7757
    Illegal BamHI site found at 3933
    Illegal BamHI site found at 5282
    Illegal BamHI site found at 8225
    Illegal XhoI site found at 963
    Illegal XhoI site found at 1137
    Illegal XhoI site found at 4606
    Illegal XhoI site found at 6952
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 3741
    Illegal SpeI site found at 8260
    Illegal PstI site found at 3437
    Illegal PstI site found at 3729
    Illegal PstI site found at 6154
    Illegal PstI site found at 6388
    Illegal PstI site found at 7600
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 3741
    Illegal SpeI site found at 8260
    Illegal PstI site found at 3437
    Illegal PstI site found at 3729
    Illegal PstI site found at 6154
    Illegal PstI site found at 6388
    Illegal PstI site found at 7600
    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 4524
    Illegal SapI site found at 5814
    Illegal SapI.rc site found at 7011


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