Part:BBa_K4621079

CRISPRi-62

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.

Usage and Biology

Fig.1 CRISPRi system used in the study

Due to the limited types of plasmids available for SCUT-3, we inserted the CRISPRi-62 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-62 system (Pi2 in the figure below) in the fermentation of ordinary LB and shrimp shells.

Fig.2 Performance test on CRISPRi strains

Fig. 3 Ectoine/hydroxyectoine production using shrimp shell waste of CRISPRi strains

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 15
  Illegal SpeI site found at 4534
  Illegal PstI site found at 3
  Illegal PstI site found at 2428
  Illegal PstI site found at 2662
  Illegal PstI site found at 3874
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4511
  Illegal SpeI site found at 4534
  Illegal PstI site found at 3
  Illegal PstI site found at 2428
  Illegal PstI site found at 2662
  Illegal PstI site found at 3874
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 467
  Illegal BglII site found at 1262
  Illegal BglII site found at 4031
  Illegal BamHI site found at 207
  Illegal BamHI site found at 1556
  Illegal BamHI site found at 4499
  Illegal XhoI site found at 880
  Illegal XhoI site found at 3226
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 15
  Illegal SpeI site found at 4534
  Illegal PstI site found at 3
  Illegal PstI site found at 2428
  Illegal PstI site found at 2662
  Illegal PstI site found at 3874
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 15
  Illegal SpeI site found at 4534
  Illegal PstI site found at 3
  Illegal PstI site found at 2428
  Illegal PstI site found at 2662
  Illegal PstI site found at 3874
  Illegal NgoMIV site found at 1824
  Illegal NgoMIV site found at 2195
  Illegal NgoMIV site found at 2398
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3406
  Illegal BsaI.rc site found at 4285
  Illegal SapI site found at 798
  Illegal SapI site found at 2088
  Illegal SapI.rc site found at 3285