Difference between revisions of "Part:BBa K5036004"

Revision as of 09:30, 11 September 2024

d-CAS9(C)

Part Description

It is a modified version of the CRISPR-Cas9 gene editing tool which cannot cut DNA. Instead, it can bind to a specific DNA sequence guided by an RNA molecule so it can be fused to transcriptional activators or repressors. In our model dcas9 was divided into N-terminal and C-terminal fragments. So the the C-terminal fragment was grafted onto NLS to generate NLS-dCas9(C) and avoid dcas9 self assembly

Usage

This part is attached to NLS and dCas9-synRTK receptor and won’t be released until cleavage of TCS1 and TCS2 This prevents the self-assembly of the two domains of dCas9 therefore providing control over the transcription activity.

literature characterization

In this study,The researchers used four strains of E. coli bacteria: DH5alpha and JM109 (both dam+/dcm+), and JM110 and GM2163 (dam-/dcm-). They introduced two plasmids, pRA-cas9 and pRA-dcas9, into each strain. These plasmids express Cas9 or dCas9 proteins under the control of a powerful, constantly active promoter called PgroESL.

(Fig.a)the presence or absence of DNA methylation (dam+/dcm+ vs. dam-/dcm-) didn't significantly affect the toxicity of Cas9 or dCas9 proteins. However, there was a slightly lower success rate in transforming dam-/dcm- strains with plasmids containing cas9/dcas9 genes compared to strains with normal methylation. This suggests that DNA methylation might play a minor role in the efficiency of plasmid transformation with these specific genes

In order to determine expression of Cas9/dCas9 in each strain of E. coli, the researcher separated the cell extracts using electrophoresis on a gel (SDS-PAGE). Finally, they visualized the proteins using two methods: staining the gel with Coomassie blue and Western blotting.

(Fig.b&c)The experiment confirmed the production of Cas9 and dCas9 proteins in all E. coli strains containing the relevant plasmids. These proteins appeared as a band around 160 kDa on the gel and were absent in strains with empty vector controls. Interestingly, the levels of Cas9/dCas9 expression were slightly higher in strains with normal DNA methylation (dam+/dcm+) compared to those lacking methylation (dam-/dcm-). Additionally, expression levels varied between strains, with DH5alpha showing the highest and GM2163 showing the lowest. Despite lower protein expression in dam-/dcm- strains, the results suggest a possibility of slightly greater toxicity associated with Cas9/dCas9 in these strains compared to dam+/dcm+ strains.