Difference between revisions of "Part:BBa K3168000"
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This part is optimized for expression of dCas9 in bacterial cells. The protein was successfully expressed in BL21(DE3) and purified with immobilized metal affinity chromatography (IMAC). All the purification steps (lysate flow through, bind flow through, wash flow through, elution) and a concentrated sample of the elution were run on a SDS-PAGE gel to evaluate the purification process. To conclude, dCas9 was successfully expressed. | This part is optimized for expression of dCas9 in bacterial cells. The protein was successfully expressed in BL21(DE3) and purified with immobilized metal affinity chromatography (IMAC). All the purification steps (lysate flow through, bind flow through, wash flow through, elution) and a concentrated sample of the elution were run on a SDS-PAGE gel to evaluate the purification process. To conclude, dCas9 was successfully expressed. | ||
IMAGE | IMAGE | ||
Revision as of 09:56, 12 September 2019
dCas9
Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease which is part of the CRISPR-immune system of bacteria such as Streptococcus pyogenes. This endonuclease is used a lot in research to facilitate efficient genome engineering (Ran, 2013). Cas9 can target any sequence by simply specifying a 20 nucleotide targeting sequencing within the guide RNA. dCas9 is a ‘dead’ variant of Cas9, because it does not have its endonuclease activity. This means that dCas9 binds dsDNA but does not cut the DNA, because of mutations in the RuvC1 and HNH nuclease domains (Park, 2017). Furthermore, this part does not include a stop codon, so fusion proteins can be made by combining parts.
Usage and Biology
dCas9 can bind to target sequences without cutting the DNA. Therefore, dCas9 can be used to recognize specific genes or unique DNA sequences. A fusion protein of dCas9 with a luciferase and/or fluorophore can be used to visualize the binding of dCas9 to dsDNA.
Expression
This part is optimized for expression of dCas9 in bacterial cells. The protein was successfully expressed in BL21(DE3) and purified with immobilized metal affinity chromatography (IMAC). All the purification steps (lysate flow through, bind flow through, wash flow through, elution) and a concentrated sample of the elution were run on a SDS-PAGE gel to evaluate the purification process. To conclude, dCas9 was successfully expressed.
IMAGE