Difference between revisions of "Part:BBa K4439013"
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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. | 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). | 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). | ||
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===Protein Purification=== | ===Protein Purification=== | ||
| + | |||
| + | <br> | ||
| + | [[File:Purification 03a.png|900px]] | ||
| + | <br> | ||
| + | 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=== | ===Cloning by PCR and KLD=== | ||
Revision as of 02:07, 12 October 2022
mSA-GFP-CBD-10xHis
Contents
Abstract
To complete
Sequence and features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Protein Characterization
Usage and Biology
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.