Part:BBa_K3883030

targeted knockdown the expression of ppc gene through CRISPRi

This part targeted knockdown the expression of ppc gene through CRISPRi to downregulate TCA cycle.By qRT-PCR, the inhibitory effect of CRISPRi system on ppc gene is verified.

Usage and Biology

Fig. 1 The pathway of pT7-dCas9-sgRNA.

The introduction of mutations in the nuclease domain of Cas9 from Streptococcus pyogenes produces nuclease defects, turning Cas9 into dCas9 protein which is deficient in nucleic acid cutting activity. But it can still target and bind certain DNA with the same precision under the guidance of the corresponding RNA. Artificial synthesis of sgRNA1(BBa_K3883004) includes a pairing region binding to gene ppc and a Cas9 handle region interacting with dCas9, so that dCas9-sgRNA1 complex can bind to target DNA elements and cause spatial block to prevent transcription extension of RNA polymerase, resulting in precise site-specific regulation of gene ppc without causing DNA damage. Through this part, we aim to inhibit the expression of gene ppc, thus inhibiting the tricarboxylic acid cycle and achieving the purpose of throttling phosphoenolpyruvate, improving the production of the project target acid.For more information, please visit our Design page:Jilin_China 2021 Design

Characterization

Characterizations of this part is in exactly the same way as BBa_K3883031.

Fig. 2 The sgRNA targeting PPCand gltA can reduce the corresponding mRNA level.(A) Primers designed for sgRNA-PPCand sgRNA-gltA. (B&C) Digestion and electrophoresis of two constructs. (D&E) The knockdown efficiency of sgRNA to PPC and gltA genes. The designed plasmid was transformed into E coli. BL21 and cultured until the value of OD600 reaches 0.4. After 8h IPTG induction, RNA extraction and reverse transcription were carried out. 16S rRNA was used as the reference gene and relative quantification was performed by qRT-PCR.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1208
  Illegal NheI site found at 4231
  Illegal NheI site found at 4254
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 49