Kaleidoscope protein ladder wiki7/14/2023 ![]() However, this band was also observed in both elution 1 and 2 (surrounded by orange box) thus indicating that the fusion protein from the soluble fragment was purified. The intensity of the band however is thicker in the insoluble fragment thus the fusion protein is majorly insoluble. In Figure 2 we can see this band in both the soluble and insoluble fragments. The TmEncH_iLOV fusion protein has a total size of around 46 kDa. From each of the samples obtained during the procedure we obtained 50 μL to use for SDS PAGE. Finally, we recycled the column ready for future purifications. Then we eluted using competitive elution by loading BXT which competed with TmEncH_iLOV for binding sites with the resin, thus detaching the protein of interest from the column. Then a washing step was performed to remove any potential non bound nonspecific proteins. The process involved packing the column, equilibrating the resin and loading the soluble sample. We proceeded on with purification of the soluble fragment using column chromatography containing Strep-Tactin resin. 50 μL from each sample were obtained and stained with Laemmli reagent. Following sonication, the sample were span to separate the soluble and insoluble fragments form the whole cell lysate. Once resuspended, the sample was cell lysed using sonication. The pellet obtained from the 50 mL cultures was then resuspended in Tris Buffer Saline at an OD600 of 10. In order to observe whether the BBa_K3111104 fusion protein was successfully expressed, we analysed our cell pellet using SDS PAGE. Then the supernatant was discarded and the pellet frozen at -80 ☌.įigure 2: SDS PAGE gel of TmEncH_iLOV L:Precision Plus Protein Kaleidoscope Protein Ladder, S: Soluble fragment, I: Insoluble fragment, Lo: Load, W: Wash, E1-2: Elution 1-2. It was spun for 10 minutes at 5000 rpm in order to pellet the cells. ![]() The culture was collected and transferred into a 50 mL falcon tube. The cultures were left to grow again overnight at 37 ☌. Once they reached OD 0.6, the cultures were induced by addition of 400 μΜ IPTG. The culture was incubated at 37☌ until it reached an OD of 0.6. Cultures were incubated at 37☌ overnight.Ī 50 mL scale-up cultures was prepared from a single starter culture containing cells carrying pSB1C3 + BBa_K3111104. Transformed colonies containing pSB1C3_ BBa_K3111104 were used to prepare overnight starter cultures containing a total of 5 mL LB broth and chloramphenicol (5 μL). Transformed cells were grown in LB agar plates containing chloramphenicol and glucose. ![]() maritima encapsulin (6-His) fused to iLOV with a StrepII-tag fusion protein. Thus, the miniprepped plasmid obtained from that colony was then transformed into BL21 (DE3) into order to proceed with 50 mL cultures to express the protein.ĭifferent batches of BL21 (DE3) competent cells were transformed with pSB1C3 plasmids containing BBa_K3111104 sequence coding for T. We expected bands at 2613 bp and 956 bp and those were obtained only for colony B as observed in Figure 1. In order to investigate whether the ligation was successful, we picked 4 colonies from the plate containing the transformed DΗ5α, grown into 5 mL cultures, miniprepped and conducted a test digest with restriction enzymes BamHI and XbaI. We started by cloning BBa_K3111104 into pSB1C3 vector. Figure 1: Test digest of BBa_K3111104 within a pSB1C3 plasmid b-d indicate repeats of different colonies containing the same plasmid cut with BamHI and XbaI type II restriction enzymes. ![]()
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