Difference between revisions of "Part:BBa K3423005"

Revision as of 13:51, 23 October 2020

a controlled protein expression system

Protein anti-CRISPR-associated 1 (Aca1) is constitutively expressed by T7 promoter, Aca1 will subsequently inhibit the anti-CRISPR promoter, so the gene expression downstream anti-CRISPR promoter will be inhibited until Navitoclax (a compound that can inhibit Aca1 found by SUSTech_Shenzhen) is added artificially. Without the inhibition from Aca1, the expression of genes downstream anti-CRISPR promoter will increase significantly. Therefore, protein expression can be regulated artificially.

When Aca1 is mutated at its 44th amino acid (Arginine to alanine, Aca1R44A),the difference expression level of green fluorescent protein could be easily observed with naked eyes:

Based on the origin composite part, we tested several candidate compounds and calculated its Ki using our model:

Then, we confirmed the effective Aca1 inhibitor Navitoclax on this composite part using green fluorescent protein as a reporter, the green fluorescence intensity is measured with a microplate reader:

At last, the binding between Navitoclax and Aca1 is identified in vitro through differential scanning fluorimetry:

Sequence and Features