Difference between revisions of "Part:BBa K3982025"

Latest revision as of 14:38, 21 October 2021

CODE M Construct C1

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 Cas14a1 protein in-vitro.

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.

Cas14 is highly specific and its miniature size makes it easy to deliver in cellular systems. It can readily form complex with sgRNA. Also, the mode of SNP detection is PAM independent, which means it does not require a PAM sequence to interact with DNA/RNA.

Plasmid Map of CODE M Construct C1. Created with SnapGene.

Methodology

Parts from Addgene #112502 are PCR amplified and cloned in pSB1C3. We propose to use this construct for tetracycline induced expression of recombinant Cas141a1. Upon addition of tetracycline, the tetracycline repressor will undergo a conformational change which makes it unable to bind to the tet operator. This will allow transcription of Cas14a1 along with the affinity tag (6x His Tag) and TEV protease i.e. Part BBa_K3982006

Sequence and Features