Composite

Part:BBa_K4621133

Designed by: Anyang Chen, Ruikang Chen   Group: iGEM23_SCUT-China   (2023-10-05)
Revision as of 11:53, 11 October 2023 by Conerrr (Talk | contribs) (→‎Usage, Biology and Characterization)

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The composite Parts of SCUT-3-GABAi9 that achieve high GABA production functiont.

Usage, Biology and Characterization

gadB is the coding sequence for glutamate decarboxylase, GAD is a pyridoxal 5'phosphate (PLP)-dependent decarboxylase widespread in bacteria yeast and filamentous fungi. The carboxyl group (-COOH) on the catalytic glutamate molecule is released to CO2, and 4-Aminobutanoate is generated at the same time, which participates in the utilization of α-Ketoglutaricacid in the TCA cycle.[1] The specific synthesis route is shown in FIG. 1. biosynthetic-pathway-for-gaba.jpg

Fig.1 Biosynthetic pathway of GABA

This CRISPRi fragment contains the dCas9 and sgRNA necessary for the CRISPRi system. The CRISPRi system is suitable for a variety of Streptomyces.[2] In this study, it was used to inhibit the expression of gabT gene in SCUT-3 to produce more GABA.

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-gadB-i9 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 and shrimp shells.

fermentation-data-of-wt-gaba-gi9.png

Fig.2 Fermentation using common LB

From the result we can observe significant improvements of GABAi9 compared to GABA and WT.

Then we conducted shrimp shell fermentation to these 3 strains.


fermentation-using-shrimp-shells-g.png

Fig.3 Fermentation using shrimp shells

From the illustration, GABAi9 still produce the highest level of GABA, with also the highest level of FAA in the fermentation broth.


Reference

[1] Guo, X. F., Hagiwara, T., Masuda, K., and Watabe, S. (2011) Biosci. Biotechnol. Biochem. 75, 1867-1871. [2] 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 SpeI site found at 5966
    Illegal PstI site found at 3860
    Illegal PstI site found at 4094
    Illegal PstI site found at 5306
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 5943
    Illegal SpeI site found at 5966
    Illegal PstI site found at 3860
    Illegal PstI site found at 4094
    Illegal PstI site found at 5306
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1899
    Illegal BglII site found at 2694
    Illegal BglII site found at 5463
    Illegal BamHI site found at 1639
    Illegal BamHI site found at 2988
    Illegal BamHI site found at 5931
    Illegal BamHI site found at 6136
    Illegal XhoI site found at 2312
    Illegal XhoI site found at 4658
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 5966
    Illegal PstI site found at 3860
    Illegal PstI site found at 4094
    Illegal PstI site found at 5306
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 5966
    Illegal PstI site found at 3860
    Illegal PstI site found at 4094
    Illegal PstI site found at 5306
    Illegal NgoMIV site found at 53
    Illegal NgoMIV site found at 1242
    Illegal NgoMIV site found at 3256
    Illegal NgoMIV site found at 3627
    Illegal NgoMIV site found at 3830
    Illegal AgeI site found at 470
    Illegal AgeI site found at 690
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 4838
    Illegal BsaI.rc site found at 5717
    Illegal SapI site found at 2230
    Illegal SapI site found at 3520
    Illegal SapI.rc site found at 4717


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