Difference between revisions of "Part:BBa K3423005"

Revision as of 14:00, 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:

Figure 1. Aca1 inhibit the expression of green fluorescent protein compared to Aca1R44A which cannot inhibit acr promoter

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

Figure 2. in vivo KI of different PDI candidates

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:

Figure 4. Efficiency of navitoclax measured with green fluorescence intensity using part BBa_K3423005

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

Fig5. Binding between navitoclax and Aca1 confirmed using DSF

Sequence and Features