Part:BBa_K3982032

CODE M Construct C4

This construct has been used in Project CODE M by Team IISER_Berhampur 2021. The aim of this project is to combat Multidrug-Resistant Tuberculosis (MDR-TB) with our diagnostic kit CODE M using mobile phone microscopy. Mutant strains of Mycobacterium tuberculosis, the causative agent of MDR-TB (which confer drug resistance towards two main drugs for TB cure - isoniazid and rifampicin) are detected through high-fidelity SNP detection using CRISPR/Cas technology. Here we used Cas14a1 (also called as Un1Cas12f1) as the RNA guided nuclease with our set of customised CODE M sgRNAs.

This construct has been assembled using various basic parts to synthesize the customized sgRNA in-vitro for use with Cas14a1 protein.

Usage and Biology

CRISPR/Cas technology has been derived from the CRISPR/Cas systems present in archaea and bacteria. They function as an adaptive immune system against invading foreign DNA and RNA. This technology has grown in the past several years with a wide range of applications. There are different types of Cas proteins such as Cas9, Cas12, Cas13 as well as Cas14. All of them are RNA guided nucleases - with a crRNA and tracrRNA or a sgRNA.

sgRNAs have significantly evolved from the natural guide RNAs which consisted of two parts - crRNA and tracrRNA. The optimized structure of sgRNA makes it highly efficient for genome editing comparable to that of spCas9 guide RNAs.

Plasmid Map of CODE M Construct C4. Created using SnapGene.

Methodology

This construct is based on lac operon. We propose to add IPTG (Isopropyl ß-D-1-thiogalactopyranoside) to the competent cells which will then release the tetrameric repressor from the lac operator in an allosteric manner. This will allow the transcription of synthetic sgRNA in the lac operon and thus the synthesis of Part BBa_K3982007.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 124
  Illegal XbaI site found at 148
  Illegal PstI site found at 160
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 124
  Illegal PstI site found at 160
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 124
  Illegal BglII site found at 598
  Illegal BamHI site found at 142
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 124
  Illegal XbaI site found at 148
  Illegal PstI site found at 160
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 124
  Illegal XbaI site found at 148
  Illegal PstI site found at 160
  
• 1000
  COMPATIBLE WITH RFC[1000]