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Microsynth single tube sequencing7/13/2023 The positive control worked also as expected (B). No plasmid were uptaken by bacteria, thus no resistance was obtained. (C) Bacteria transformed with our 01a plasmid onto a Kanamycin plate.Īnalysis The negative control worked as expected (A). Bacteria were transformed with a standard GFP-plasmid. (B) Positive control onto an Ampicillin plate. (A) Negative control onto a Kanamycin plate. The plates were incubated at 37☌ overnight. All the steps were performed under sterile conditions (with a flame). Results Figure 2 Bacterial transformation plates. Since the bacteria will be treated with Kanamycin, we expect to observe grown colonies from the 01a transformed plate and the positive control since the plasmid contains a gene resistant to Kanamycin, and the absence of colonies with the negative control, since there is no antibiotic resistance. We used a GFP plasmid from the summer school as a positive control. We used water as negative control to model the absence of plasmid. Silk Fusion Protein Protein expression and purification - Trial 1Īim To do the transformation of BL21(DE3) cells with the 01a plasmid (mSA-silk-CBD). Therefore we will research another method to avoid the cellulose back step. Indeed, the latest may be a problem for the dialysis step, since we put the proteins in a cellulose membrane. For the silk protein, we should change some steps of this purification procedure because of the solubility tag and of the CBD tag. This was a good training of the protocole we are going to use for the protein purification of our SR proteins. The last two elution samples contained the least amount of protein, which was expected as well.Ĭonclusion Overall, we successfully purified the BirA protein. Then, as expected, we obtained the biggest amount of proteins in the first 4 elution samples, that is why we selected them for the dialysis. Since the protein appears to be produced in large quantities by E.Coli, we even find them in the two last washes. By analysing the results of the SDS-PAGE we can clearly see a band around 30kDa in almost all the samples which confirms the presence of the protein. We obtained a concentration c = 2.11 g/L, and a final protein volume of 17 mLĪnalysis By using the Beer-Lambert law, we obtained a final protein concentration of 2.11 g/L after the purification and the dialysis. Table 2 Absorbance measured using NanoDrop Trials The biggest amounts of our target protein can be seen in columns Elution 1-1, 1-2, 1-3 and 1-4. After having done the purification, we obtained fractions that we ran on a SDS-PAGE gel using a 4x LDS loading dye. Results Figure 1 SDS-PAGE of the different purification fractions. The first 4 elution samples were dialyzed overnight in 300 mM NaCl and 20mM HEPES 7.5 From the results we will decide which fractions to pool (combine) and add protease (if tag removal is required). This is because until we see the band on a gel we have no idea where the protein is. This includes the lysate, supernatant, all the washes and all the elutions. ![]() We will run all the fractions on a gel, so solutions from every step. Protocol Protein purification PTPSP and SDS-PAGE We did an affinity purification with Ni-NTA beads since the BirA contained a His tag, as well as our engineered proteins. For this training, we purified BirA proteins that were already induced with IPTG in E.Coli (strain K12). Indeed, upon seeing our design, the protein facility offered us the opportunity to train in protein purification, so that we would be ready for the purification of our designed proteins. Aim To train ourselves for protein purification.
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