Part:BBa_K5096071

sgRNA 70 inhA

The sequence encodes the complete sgRNA70, which guides the dCas9 protein to a specific genomic site. In this case, sgRNA70 targets the inhA region of the M. tuberculosis genome. The binding sequence of sgRNA70 is derived from the inhA gene, chosen for its low mutation rate and essential role in the pathogenicity and survival of M. tuberculosis. The inhA gene encodes NADH-dependent enoyl-acyl carrier protein reductase, a crucial enzyme in mycolic acid synthesis, a vital component of the bacterial cell envelope of M. tuberculosis (Marrakchi et al., 2014). By inhibiting mycolic acid synthesis through the CRISPRi system, sgRNA70 seeks to destabilize the bacterial cell wall, thereby weakening the pathogen. The CRISPR sgRNA design tool from Benchling was utilized to generate the guide sequence, along with the sgRNA-tracr and sgRNA repeat from Marshall et al. (2018). To validate the efficacy of sgRNA70, repression of the target inhA construct was tested in a TXTL cell-free system. After calculating the percent repression, sgRNA70 demonstrated a 60.4% reduction in fluorescence compared to the positive control. This significant decrease indicates that sgRNA70 effectively downregulates the inhA gene, potentially disrupting the pathogenicity of M. tuberculosis and improving efforts to combat the bacteria.

Lambert iGEM also utilized MATLAB to perform precise modeling of gene expression of inhA, a gene linked to the pathogenicity of M. tuberculosis (see CRISPRi M. tb POC for more about inhA). We created a complete set of ODEs, and then used Simbiology’s Model Analyzer to simulate the concentration of GFP over 20 hours with 1 nanomole of the inhA DNA initially. As we are producing GFP protein, the graph will increase at first, and then around four hours, the graph will plateau.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 36
  Illegal NheI site found at 59
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]