Part:BBa_K1585350:Experience

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Applications of BBa_K1585350

The construct is part of a two-plasmid knockout system published by [http://aem.asm.org/content/81/7/2506.long| Jiang et al]. This part, the pTargetT, contains the sgRNA to target the glgX coding sequence. Additionally, the construct consists of a repair template with two 400 bp homology arms for homology-directed repair. To succesfully knock out the glgX, you need a second plasmid, pCas. It contains the pCas gene as well as a temperature-sensitive repA101ts ori. To increase recombination efficiency in E. coli, it also expresses lambda-Red genes behind an inducible arabinose-promoter. Upon induction with IPTG, a sgRNA is transcribed to cut the pMB1 ori of the pTargetT.

After cloning both the targeting plasmid and the pCas plasmid in BL21 Gold (DE3) and inducing with arabinose, the Cas9 gene is expressed. Guided by the sgRNA, it cleaves the double strand at the beginning of the glgX gene. The succesful genome editing could be verified by sequencing.

Genomic region and sequencing chromatograms of BL21 Gold (DE3) ΔglgX The sequencing results show that the "IGEM AACHEN MULTISTOP" sequence disrupts the gene. This insert contains a stop codon in each possible frame. The picture demonstrates the functionality of BBa_K1585350.

User Reviews

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