Part:BBa_K4439013

mSA-GFP-CBD-10xHis

Abstract

To complete

Sequence and features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Protein Characterization

Modeling

We also designed a control to prove the effective attachment of our recombinant proteins.

Figure 9 : AlphaFold2 prediction for 03a. (A) First rank 3D model prediction of 03a protein; the iteration who got the highest score in the modeling. (B) Different correlation graphs between the query sequence of 03a and the predicted one, for each proposed 5 models. (C) Figure of sequence coverage of 03a and indices on alignment with other sequences in the mSA. (D) IDDT graph for 03a per residue to get an idea of the confidence in the model in predicting.

• Analysis

In (fig.9, A), we could identify the three main parts of the mSA-GFP-CBD protein. Compared to the previous (fig.9, A) we could see that the GFP domain, in green, is more easily characterized and would yield a closer linkage to the mSA chain, in blue. So we will have both of the proteins closely associated. However for the CBD region, we identified a longer linkage which would mean different configuration of tha attachment possible. From (fig.9, B, D) it was really interesting to see that the 5 predictions yielded similar correlation measures and similar IDDT scores, which enabled us to confirm that the model is robust in the determination of three chain structures. The linkage would give in reality more freedom of placement of those chains.

Results

Bacterial Transformation

We performed this bacterial transformation following the E.coli Competent Cells Quick Protocol FB035 from Promega. We transformed BL21(DE3) cells with our pET28a plasmid backbone containing mSA-GFP-CBD-10xHis coding sequence.

Figure 1 | Bacterial transformation with the 03a construct in BL21(DE3) competent cells. Remarks: unfortunately we got rid of the Rosetta plate and the control plates thinking that we took a picture of them, but it was not the case, so we do not have a picture of the empty plate and transformation controls.

• Analysis

After the overnight incubation, we observed the presence of colonies only in the BL21(DE3) transformed cells plate, but not in the Rosetta transformed cells plate. The negative control plate did not show any colony, and the positive control plate was full of colonies, as expected. The bacterial transformation of 03a worked for BL21(DE3) strain, so we can pursue the process by doing colony picking in a sterile environment (with a flame).