Project

Part:BBa_K3982030

Designed by: Akankshya Ramkrishna Sahu   Group: iGEM21_IISER_Berhampur   (2021-10-01)
Revision as of 11:56, 14 October 2021 by Akankshya16 (Talk | contribs)


CODE M Construct C2

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


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]


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Categories
Parameters
None