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).

Protein Purification

Figure 2. Protein purification of the GFP fusion protein (03a). (A) SDS-PAGE gel stained with Coomassie Blue Protein Stain of all the fractions of GFP fusion protein purification (B) Western Blot of the elution 1 fraction (E1 on SDS-PAGE) visualised with anti-His antibody. W1 = Wash 1 with 20 mM imidazole; W2 = Wash 2 with 50 mM imidazole; E1 = Elution 1 with 250 mM imidazole; E2 = Elution 2 with 500 mM imidazole; E3 = Elution 3 with 1M imidazole; E4 = 2.5 M imidazole; E5 = 5 M imidazole.

• Analysis

On both the SDS-PAGE and the Western Blot for the protein purification of the GFP fusion protein (03a) (fig 2), we observed a band around 70kDa which matched the expected size of our protein (71.4 kDa). Purified GFP fusion protein (03a) was present in all eluted fractions. To concentrate the proteins, we used a 30 kDa filter. However, the filter was made of cellulose, so we observed the filter turning green. This showed the efficiency of the CBD domain of our construct to bind cellulose. We finally obtained a concentration of 0.35 mg/mL, which corresponds to 4.5 mg of GFP fusion proteins. Since high imidazole concentration might denature the proteins, we had to either remove it or make imidazole inert. It was not possible to dialyse the proteins to remove imidazole, since the dialysis membrane was made of cellulose, and our fusion proteins would all bind to the membrane via their cellulose binding domain (CBD). We therefore decided to flash freeze the proteins and store them in the elution buffers so the imidazole will no longer affect them when frozen.

Cloning by PCR and KLD

The elution of the proteins of interest in such high amounts of imidazole certainly comes from the double His-tag that composes our proteins. To remove it from the received plasmids, we performed some cloning experiments to obtain better yields in the future purifications. In the case of the 03a plasmid (containing GFP) and the 01b plasmid (containing SR), we performed KLD cloning. We amplified the plasmids by PCR without the undesired sequence, and we re-ligated them back by doing a KLD. The KLD reaction allows efficient phosphorylation, intramolecular ligation and template removal in a single 5-minute reaction step at room temperature.

Figure 5: Cloning experiment results for removal of the added site for 01b (SR) and 03a (GFP). (A) Agarose gel electrophoresis of PCR products of the 01b and the 03a constructs plasmid amplified without the GeneScript additional tags for KLD cloning. (B) Agarose gel electrophoresis for restriction analysis of the 01b and the 03a constructs plasmid after the ligation by KLD. (C) Plasmid map from the sequencing result of the obtained new SR plasmid. (D) Plasmid map from the sequencing result of the obtained new GFP plasmid.

• Analysis

On the agarose gel we ran with the PCR products (fig 5.A), we observed bands around 7000 bp which are the expected sizes and means that we successfully removed the added site. After the KLD, we ran a second agarose gel with fragments obtained by cutting the new plasmids with different restriction enzymes (fig 5.B). We obtained similar patterns as the ones expected. By purifying and sequencing the plasmids(fig 5.C & D), we obtained the exact same sequence as designed. This confirmed that we removed the added site and re-ligated the DNA to obtain the good plasmids.

With several minipreps we obtained big quantities of these new plasmids. We therefore transformed new BL21(DE3) E.Coli competent cells to start the protein production. For the GFP fusion protein, the pellet after the first centrifugation step was not green, which means that there was no expression of our protein of interest. In a similar way, we couldn’t see a significant protein expression after the IPTG induction for the SR fusion protein. Even if the purification was performed, nothing would be visible in the elution lanes of the SDS-PAGE which suggests that we didn’t successfully purify our new SR fusion protein.

The cloning with PCR amplification and KLD for ligation was a success, but the bacteria were not able to produce our fusion proteins. Our hypothesis is that the number of bp between the RBS and the first Methionine was not optimal for the bacteria. However, by lack of time, we decided not to troubleshoot the reasons for this failure, and we concentrated on the protein production to use them in our further experiments.