Part:BBa_K4621080

CRISPRi-294

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-294 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-294 system (Pi4 or ProPi4 in the figure below) in the fermentation of ordinary LB, high-salt 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 30
  Illegal SpeI site found at 4549
  Illegal PstI site found at 18
  Illegal PstI site found at 2443
  Illegal PstI site found at 2677
  Illegal PstI site found at 3889
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4526
  Illegal SpeI site found at 4549
  Illegal PstI site found at 18
  Illegal PstI site found at 2443
  Illegal PstI site found at 2677
  Illegal PstI site found at 3889
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 482
  Illegal BglII site found at 1277
  Illegal BglII site found at 4046
  Illegal BamHI site found at 222
  Illegal BamHI site found at 1571
  Illegal BamHI site found at 4514
  Illegal BamHI site found at 4719
  Illegal XhoI site found at 895
  Illegal XhoI site found at 3241
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 30
  Illegal SpeI site found at 4549
  Illegal PstI site found at 18
  Illegal PstI site found at 2443
  Illegal PstI site found at 2677
  Illegal PstI site found at 3889
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 30
  Illegal SpeI site found at 4549
  Illegal PstI site found at 18
  Illegal PstI site found at 2443
  Illegal PstI site found at 2677
  Illegal PstI site found at 3889
  Illegal NgoMIV site found at 1839
  Illegal NgoMIV site found at 2210
  Illegal NgoMIV site found at 2413
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3421
  Illegal BsaI.rc site found at 4300
  Illegal SapI site found at 813
  Illegal SapI site found at 2103
  Illegal SapI.rc site found at 3300