MolecularCloud July Newsletter: Multicopy Chromosomal Integration Using CRISPR-Associated Transposases

Controlling the copy number of gene expression cassettes is an important strategy to engineer bacterial cells into high-efficiency biocatalysts. Current strategies mostly use plasmid vectors, but multicopy plasmids are often genetically unstable, and their copy numbers cannot be precisely controlled. The integration of expression cassettes into a bacterial chromosome has advantages, but iterative integration is laborious, and it is challenging to obtain a library with varied gene doses for phenotype characterization.

Dr. Sheng Yang’s team published the paper “Multicopy Chromosomal Integration Using CRISPR-Associated Transposases” in ACS Publications on June 17, 2020. All plasmids of this article have been deposited on MolecularCloud. 

In this paper, Dr. Yang’s team demonstrated that multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) can be achieved by designing a crRNA to target multicopy loci or a crRNA array to target multiple loci in the Escherichia coli genome. Within 5 days without selection pressure, E. coli strains carrying cargos with successively increasing copy numbers (up to 10) were obtained. Recombinant MUCICAT E. coli containing genomic multicopy glucose dehydrogenase expression cassettes showed 2.6-fold increased expression of this important industrial enzyme compared to E. coli harboring the conventional protein-expressing plasmid pET24a. Successful extension of MUCICAT to Tatumella citrea further demonstrated that MUCICAT may be generally applied to many bacterial species.

Plasmids from this article in MolecularCloud

Cat. No.

Plasmid Name

Description

MC_0101094

pQCascade

pCDFDuet-1 harboring TniQ, Cas8, Cas7 and Cas6 from V. cholerae strain HE-45 and inserting BsaI restriction sites flanked by two CRISPR repeats

MC_0101095

pQCascade-array1

Multi-copy chromosomal integration by CRISPR-associated transposases in Escherichia coli

MC_0101096

pQCascade-IS1

Multi-copy chromosomal integration by CRISPR-associated transposases in Escherichia coli

MC_0101097

pQCascade-IS2

Multi-copy chromosomal integration by CRISPR-associated transposases in Escherichia coli

MC_0101098

pQCascade-IS3

Multi-copy chromosomal integration by CRISPR-associated transposases in Escherichia coli

MC_0101099

pQCascade-IS186

Multi-copy chromosomal integration by CRISPR-associated transposases in Escherichia coli

MC_0101101

pDonor-GDH

Multi-copy chromosomal integration by CRISPR-associated transposases in Escherichia coli

MC_0101100

pQCascade-tra13

Multi-copy chromosomal integration by CRISPR-associated transposases in Escherichia coli

MC_0101102

pSC101-Donor

Multi-copy chromosomal integration by CRISPR-associated transposases in Escherichia coli

MC_0101103

pHelper-T7IS1

Multi-copy chromosomal integration by CRISPR-associated transposases in Escherichia coli

MC_0101104

pCutamp

Plasmid-curing in Escherichia coli by targeting the AmpR promoter



These plasmids will be free plasmids for August as exclusive benefits for MolecularCloud members. Leave a comment and share your research result about CRISPR(no less than 100 words). Get one of August designated plasmids for free!


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