Part:BBa_K3423005
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 3. 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):
Figure 4. Binding between navitoclax and Aca1 confirmed using DSF
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1057
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