Once we had our idea settled and AbDEN was conceived, our wet lab team buckled down and got to work. To successfully fulfill our plans, we conducted various experiments throughout the duration of our project. Our lab work started with our summer break and continued till the wiki freeze, and even beyond. A week-wise summary of what we did in our lab during this iGEM cycle to bring AbDEN to life is given below.
May
Week 4
This week, we were taught the procedure of Agarose Gel Electrophoresis by our Instructor, Ekta Gupta. We initially ran some of her samples in the gel and then performed the procedure ourselves. We shared the results of the experiment with our instructor too.June
Week 1
We learned how to make buffers such as acid-salt buffer (ASB) and phosphate-buffered saline (PBS). We also learned how to prepare culture media like the Luria Bertani agar and Luria Bertani broth. We then practised streaking E. coli DH5-ɑ cells.Week 2
We learned how to make primary cultures of E. coli DH5-ɑ cells, perform optical density measurements at 600 nm (OD600), and then make a secondary culture by inoculating the primary culture into Luria Bertani broth. We also performed the chemical competency protocol for the E. coli DH5-ɑ cells.Week 3
We performed the transformation protocol of the E. coli DH5-ɑ cells using some plasmid vectors provided by our instructor, Ashwin Uday. We received the SHuffle strains of E. coli and some plasmids, including cIgG-pET21b-cyclonal, on 16th June from our Secondary PI, Dr. Mehmet Berkmen.Week 4
We streaked the SHuffle cells received, made glycerol stocks of them, and then stored them at -80℃. We also performed plasmid isolation to extract the plamids from their host cells, and stored them at -20℃.July
Week 1
We performed the protein expression and expressed anti-Maltose Binding Protein IgG (aMBP-IgG) in SHuffle B cells. For this, we had to culture SHuffle B cells and make them competent. Then we had to transform the aMBP-IgG plasmid vector into the competent cells.Week 2
We performed our first SDS-PAGE using some proteins provided to us by our instructor, Ashwin Uday. We also sonicated the SHuffle B cells containing the aMBP-IgG expressing plasmid vector.Week 3
We performed SDS-PAGE on our expressed aMBP-IgG antibodies. We then purified the antibodies using protein A chromatography and ran another SDS-PAGE on the purified antibodies. We also received the anti-maltose binding protein IgY (aMBP-IgY) sequence from Integrated DNA Technologies (IDT).Week 4
Repeated chemical competency for the K-12 cells. We transformed aMBP-IgY plasmid into DH5α cells with these plasmids. We lysed the cells, extracted the plasmid to use for cloning. We cultured SHuffle cells containing the aMBP-IgG plasmids under different IPTG concentrations to determine the optimum concentration. We sonicated the cells grown under different conditions and performed SDS-PAGE to determine the best concentration of IPTG.Week 5
Made further attempts at cloning the IgY sequence. We also performed another round of purification for the aMBP-IgG antibodies and then ran the purified antibodies on an SDS-PAGE.August
Week 1
We analyzed all the colonies we got after transforming with the cloned plasmid and noticed that the ligation control had more colonies than the 1:3 plate. We checked the efficiency of the cloning through alkaline lysis. We expressed aMBP-IgG once again in a larger SHuffle culture for better purification.Week 2
The SHuffle cells containing the aMBP-IgG plasmid vector were sonicated to extract the antibodies from the cells, and protein A chromatography was done to purify the aMBP-IgG antibodies. We also performed a confirmatory restriction digest on the positive clones we had obtained after the second round of cloning the aMBP-IgY sequence into a pET21b vector and ran the digest on a gel.Week 3
We obtained a positive clone of the aMBP-IgY sequence inserted in a pET21b vector. We then transformed this plasmid into competent Shuffle B cells and even sent it for sequencing analysis for final confirmation. The bacteria were grown in different conditions and then sonicated to extract the expressed antibodies. An expression SDS-PAGE was run on all the samples. We also ran an SDS-PAGE on the purified IgG samples. We started the ultracompetency procedure for the SHuffle-K12 cells and made buffers for Ni-NTA chromatography. We also received the C10-IgG antibody sequence in a pUC vector from IDT, which was transformed into E. coli DH5-ɑ.Week 4
This week, we completed the ultracompetency procedure for the SHuffle K-12 cells. We also performed the miniprep plasmid extraction procedure on the E. coli DH5-ɑ containing the C10 sequence. We received the scFv sequences from IDT. When we set up the restriction digest for the C10 scFv, the digestion was partial. Hence, we concluded that the vector was untrustworthy and the insert was insufficient for ligation.Week 5
We transformed all the vectors containing the scFv sequences into E. coli DH5-ɑ. We then cultured these cells and then isolated the plasmid from them. Miniprep procedure was conducted for two E. coli DH5-ɑ cultures containing different scFv sequences, empty and mid, and two pET21b empty plasmids. We also set up a restriction digest for the E. coli DH5-ɑ culture containing the empty scFv sequence, which was successful. Then, we set up the ligation process (V, V+lig, 1:3) and transformed the ligation mixture. The ligation was successful as well. We then made 10 primary cultures from the ligation plate. Meanwhile, the sequence coding for IgY present in the pET21b vector was transformed once again into competent SHuffle cells. After some time, when the cells had expressed the plasmid, they were sonicated, and Ni-NTA chromatography purification was done on the supernatant received after sonication.September
Week 1
We performed SDS-PAGE on the purified IgY antibodies. We observed bands at the right place, but there were multiple bands, maybe because of fragmentation. We also did an alkaline lysis check for the positive clones of the E. coli DH5-ɑ cells containing the empty scFv sequence. The positive clones were then transformed into SHuffle B cells. We also began the design of experiments (DoE) process by calibrating the results of the SDS-PAGEs with BSA standards.Week 2
We made glycerol stocks of the SHuffle cells transformed with the scFv sequences. We also made the lysis buffer required for sonication. The E. coli DH5-ɑ cells containing the empty scFv sequence expressed the protein and were pelleted down.Week 3
The pellets of the E. coli DH5-ɑ cells containing the empty scFv sequences were sonicated. The supernatant was then purified and run on SDS-PAGE. We set the scFv-linker restriction digest. The ligation check showed insufficient insert DNA.Week 4
We continued the DoE experiments. We repeated the procedure for the scFv-linker restriction digest. When the gel ran and the insert DNA was extracted, we concluded that the ligation setup was successful. We performed alkaline lysis for the positive clones. We also conducted a confirmatory digest on the scFv-linker with NdeI and XhoI and ran it on a gel. The positive clones were then transformed into competent SHuffle cells.October
Week 1
We made the primary cultures and the subsequent glycerol stocks of the SHuffle cells transformed with the sequence for the scFv-linker. One of the positive clones was used to inoculate a 100 mL secondary culture. The culture was then pelleted down and sonicated. The cultures transformed with the scFv sequences were grown in 3 conditions: 16℃, 25℃, and 30℃. All the cultures were pelleted down and sonicated. All the scFv-blank conditions and the scFv-linker were run on an SDS-PAGE, and subsequently, a western blot was performed with anti-His antibodies. The scFv-linker and the scFv-empty protein expressed at 16℃ were purified and run on an SDS-PAGE. All 15 of the DOE experiments were lysed, the OD of the resuspended cells were somewhat normalized, and the supernatant was run on triplicate SDS-PAGE gels, which were used for statistical analysis.Week 2
Purified proteins were concentrated with the help of a centricon, flash-freezed and stored at -80 ℃. NeoFV was transformed into SHuffle B, SHuffle K-12 and BL-21 cells and the cultures were grown for 12 hours to obtain a growth curve. A thermal shift assay was performed with an aliquot of the purified proteins. Some aliquots were shipped to Rahul Roy in IISc, Bangalore to perform VLP Fusion Assay.
The protocols behind the experiments performed by us as part of our wet lab can be found on our Experiments Page.
The specifics of the weekly experiments mentioned above can be found in the Rough Copy of our wet lab notebook.