Difference between revisions of "Part:BBa K4621082"

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===Usage and Biology===
 
===Usage and Biology===
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https://static.igem.wiki/teams/4621/wiki/parts/mechanism-of-crispri.png
 
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Fig.1 CRISPRi system used in the study
 
  
 
Due to the limited types of plasmids available for SCUT-3, we inserted the CRISPRi-239 fragment into the previously constructed plasmid by homologous recombination to achieve simultaneous stable expression of multiple target genes. Subsequently, we verified the effectiveness of the CRISPRi-239 system in the fermentation of ordinary LB and shrimp shells.
 
Due to the limited types of plasmids available for SCUT-3, we inserted the CRISPRi-239 fragment into the previously constructed plasmid by homologous recombination to achieve simultaneous stable expression of multiple target genes. Subsequently, we verified the effectiveness of the CRISPRi-239 system in the fermentation of ordinary LB and shrimp shells.
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https://static.igem.wiki/teams/4621/wiki/parts/fermentation-data-of-wt-gaba-gi9.png
 
https://static.igem.wiki/teams/4621/wiki/parts/fermentation-data-of-wt-gaba-gi9.png
  
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Fig.2 Performance test of GABA over-produced strains
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Fig.1 Performance test of GABA over-produced strains
  
 
https://static.igem.wiki/teams/4621/wiki/parts/fermentation-using-shrimp-shells-g.png
 
https://static.igem.wiki/teams/4621/wiki/parts/fermentation-using-shrimp-shells-g.png
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Fig.3 GABA production using shrimp shell waste of genetically engineered SCUT-3
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Fig.2 GABA production using shrimp shell waste of genetically engineered SCUT-3
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===Reference===
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[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.
  
  

Latest revision as of 12:11, 11 October 2023


CRISPRi-239

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 gabT gene in SCUT-3 to produce more GABA.


Usage and Biology

Due to the limited types of plasmids available for SCUT-3, we inserted the CRISPRi-239 fragment into the previously constructed plasmid by homologous recombination to achieve simultaneous stable expression of multiple target genes. Subsequently, we verified the effectiveness of the CRISPRi-239 system in the fermentation of ordinary LB and shrimp shells.

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

Fig.1 Performance test of GABA over-produced strains

fermentation-using-shrimp-shells-g.png Fig.2 GABA production using shrimp shell waste of genetically engineered SCUT-3


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 SpeI site found at 4530
    Illegal PstI site found at 2424
    Illegal PstI site found at 2658
    Illegal PstI site found at 3870
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4507
    Illegal SpeI site found at 4530
    Illegal PstI site found at 2424
    Illegal PstI site found at 2658
    Illegal PstI site found at 3870
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 463
    Illegal BglII site found at 1258
    Illegal BglII site found at 4027
    Illegal BamHI site found at 203
    Illegal BamHI site found at 1552
    Illegal BamHI site found at 4495
    Illegal BamHI site found at 4700
    Illegal XhoI site found at 876
    Illegal XhoI site found at 3222
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 4530
    Illegal PstI site found at 2424
    Illegal PstI site found at 2658
    Illegal PstI site found at 3870
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 4530
    Illegal PstI site found at 2424
    Illegal PstI site found at 2658
    Illegal PstI site found at 3870
    Illegal NgoMIV site found at 1820
    Illegal NgoMIV site found at 2191
    Illegal NgoMIV site found at 2394
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3402
    Illegal BsaI.rc site found at 4281
    Illegal SapI site found at 794
    Illegal SapI site found at 2084
    Illegal SapI.rc site found at 3281