Team:CCA San Diego/Notebook
Our team mainly worked in the lab during the fall of 2021, with only four members physically conducting wet lab research at the J. Craig Venter Institute. We included a timeline of brief descriptions of what we did each week in the lab, as well as a more detailed lab notebook with our daily procedures, pictures, and results.
July 25 - July 31: Linearizing our Modified Backbone Fragment
We ran PCR on our pRSII16 plasmid to linearize our desired backbone fragment free of the lacZalpha gene. The linearized plasmid was tested in a gel alongside a ladder and the original backbone fragment to verify if the amplification was successful. The following day, three 50 uL PCR aliquots were prepared with backbones, resulting in three more linearized backbone solutions. The three solutions were also tested on the gel electrophoresis bed and showed successful PCRs. We performed a DpnI digest on each of the three PCR aliquots and the initial one from the previous day to destroy any circular backbone plasmids.
August 1 - August 7: Collagen Gelification
Our goal for this week was to make a collagen gel out of the collagen powder we purchased. This would then serve as a proof of concept for our end goal: making bacterial collagen products. Initially, our collagen powder was 2% w/v, the molarities of our coagulation buffer were most likely inaccurate, and we also neglected prior incubation of the buffer to 37°C. These factors may have led to the failed coagulation when we put the acidic collagen solution in the neutral coagulation buffer. Surprisingly, the collagen did not congeal even after incubating it with 5 mL of 5.33%, 2%, and 1% glutaraldehyde solutions for 72 hours at 37oC. Glutaraldehyde is one of the strongest cross-linkers used in the literature, which led us to suspect the problem was with the collagen powder itself. For the rest of the week, we attempted to troubleshoot our previous experiments with different procedures and buffers - instead of using a fiber formation buffer from research gate, we used a PEG coagulation buffer from Cavallaro et. al, ___. Even so, the collagen did not coagulate. These results reinforced the notion that the collagen powder itself was the culprit. To test this, we obtained two 2.4 mg of bovine cartilage containing approximately 1.44 mg of Type I collagen. Since it was unable to dissolve in the acetic acid solution after intense vortexing, we let it dissolve in acetic acid for 72 hours. Additionally, we tested three different high-concentration solutions of our collagen powder so we could simultaneously test the variable of concentration.
August 8 - August 15: Porcine Collagen Gelification
For this week, we continued our attempt at making gel. Due to failures from previous tests of concentration and source failure (a result of the impure collagen powder), we decided to shift to a new procedure: porcine collagen gelification for the purpose of deriving pure collagen. We used pig skin from a local butchery, using a porcine extraction procedure to obtain collagen. Since the powder that we had previously used in our gels had not worked, we hoped this would. Another important component in our collagen gelification attempts was determining the correct amount of crosslinking reagent to use. We conducted gelification trials with varying glutaraldehyde concentrations and various buffers: water, PEG coagulation buffer, and fiber formation buffer. We placed agitating collagen samples in petri dishes of the 3 buffers, and the collagen samples in the PEG coagulation buffer ended up the least dispersed and the most coagulated. This made PEG the best buffer for our purposes. We tried a different gel procedure that utilised pepsin, which was also unsuccessful. Therefore, we believe that the collagen powder and porcine collagen are at fault. When our gelification attempts failed for the porcine collagen, we ran an SDS page to verify the purity and presence of collagen proteins from our porcine collagen extraction. Our SDS page results do not correspond to the expected results. The collagen powder wells had the thickest bands, with double bands around 120kDa — the telopeptide-positive collagen had a semi-faint band around 120kDa. Meanwhile, we began preparations for next week's procedures. We intend to run Gibson assembly and yeast assembly in parallel for the coming week. We successfully made numerous E.coli-based agar plates and yeast-based agar plates. This will be necessary for the procedures next week by providing means of growing URA3 mutant strain yeast and E.coli.
August 16 - August 21: 2nd Iteration of Porcine Gelification
Our initially planned Gibson and yeast assemblies were delayed because the DNA shipment was cancelled. We maintained our focus and approached collagen gelification through a new procedure. The new method involved a chemical separation of pig skin mass through numerous acid baths. When purifying the collagen protein, we introduced samples with pepsin to compare the results of telopeptide-rich and -poor proteins. After pelleting cycles were completed, the collagen samples appeared to have salted out proteins as a precipitate. To confirm this, we ran an SDS page on telopeptide-rich, telopeptide-poor, and pure porcine samples, as well as our old hydrolyzed collagen and bovine collagen. The results were troubling, as they not only did not indicate any significant trend, but also were inconsistent with our previous SDS pages on bovine and hydrolyzed collagen. This suggests a problem with the SDS page itself, so we will continue with the gelification procedure.
August 23 - August 27: DNA
This week, we worked with our newly arrived DNA. On August 23rd, we amplified the necessary primers needed. Furthermore, we worked on back-diluting the yeast in order to prepare it. After our DNA arrived, we amplified them via PCR and ran both Gibson and yeast assemblies in parallel in order to create our plasmid. After running the Gibson and the yeast assembly, we conducted an E. Coli transformation for self-closure and ran another PCR amplification. Afterwards, we noticed that our colonies were not growing after two days, and this phenomenon only occurred in the Amp (-) plates. We opted for a new yeast and Gibson assembly. These were incomplete and will be completed on August 30.
August 30 - September 4: DNA
After finishing both the yeast and Gibson assembly, we ran an SDS page to see the results of the collagen gels and the bacterial transformation. There were no bands seen for the E. coli, but there were prominent bands visible for the collagen gels. The next day it was determined that the E. coli from the Gibson grew colonies, and a mini prep procedure was performed. We experimented with a new procedure for the gels, which showed successful gelification within 30 minutes of execution. However, it did not maintain a gel-like form for a 24 hour period. We will conduct further experimentation with the cosolvent ratios and the collagen ratios to find the optimal ratio for the gels. September 7 - September 12: Troubleshooting and Collagen Gelification Experimentation We performed a Gibson assembly transformation and implemented PCR to amplify our gene fragments (for future use in Gibson and yeast assemblies). To troubleshoot what may have went wrong in last week's Gibson and yeast assemblies (the gel we did after the miniprep from last week showed no bands), we conducted E. coli transformation on the following: We used our PRS backbone as a positive control, last week's Gibson assembly products and no DNA as a negative control, and cells from last week's miniprep on a carbenicillin plate. Colonies grew on the carbenicillin plate, so we prepared for the miniprep by growing out cells in LB medium and amp. We then did miniprep to purify our plasmid. We further experimented on the new procedure for collagen gels involving a citrate buffer by changing the ratios of glycerol to collagen protein. Changes in collagen protein concentration showed a negligible change. Glycerol concentration proportionally increased viscosity of the sample, which, however, showed no indications of coagulation.
September 7 - September 12: Troubleshooting and Collagen Gelification Experimentation
We performed a Gibson assembly transformation and implemented PCR to amplify our gene fragments (for future use in Gibson and yeast assemblies). To troubleshoot what may have went wrong in last week's Gibson and yeast assemblies (the gel we did after the miniprep from last week showed no bands), we conducted E. coli transformation on the following: We used our PRS backbone as a positive control, last week's Gibson assembly products and no DNA as a negative control, and cells from last week's miniprep on a carbenicillin plate. Colonies grew on the carbenicillin plate, so we prepared for the miniprep by growing out cells in LB medium and amp. We then did miniprep to purify our plasmid. We further experimented on the new procedure for collagen gels involving a citrate buffer by changing the ratios of glycerol to collagen protein. Changes in collagen protein concentration showed a negligible change. Glycerol concentration proportionally increased viscosity of the sample, which, however, showed no indications of coagulation.
September 13 - September 18: Yeast Assembly and Collagen Gelification with Neutral pH
The yeast grew on their plates, but were concentrated into clumps with dead cells. As a result, we had to do a patch on fresh plates to ensure the yeast could grow. However, the negative control without any DNA also grew on the URA- plates, indicating that either our plates or the yeast cells had a problem with them. After we did the patch, we noticed similar results, despite purifying protein lysates and DNA from the yeast. Running an SDS-PAGE on the DNA showed nuclear bands, likely due to a low concentration or too much chromosomal DNA. Thus, we did another yeast assembly with just the formed plasmid from Gibson assembly, but no colonies grew on our plates. As we got Sanger sequencing results showing faulty bp alignment, this may indicate that our initial Gibson assembly did not work. For collagen gelification, we took a step back in our approach and revised our previous PBS mix gel. We tested specifically for neutral pH's effect via NaOH balancing and received promising results; the gels coagulated instantly in thicker, more condensed blocks. The masses were more opaque and had a distinctive white color. Few tiny particles broke off until the gel had been in solution for an extended period of time. We concluded that changing the pH to neutral was the solution for strong gelification but pH testing on the citrate gels showed no response.
September 20 - September 26: DNA and Implementations
DNA: This week we made new plates for our yeast assembly with various amino acids, excluding uracil, yeast nitrogen base, agar, and dextrose. We made new plates because yeast colonies were growing on our URA3(-) plates, which shouldn't have been happening, therefore we concluded that our plates were old and contaminated with uracil. We did a Gibson assembly with both 2 fragments and 3 fragments and transformation again, and yeast assembly transformation. After transformation, we grew yeast cells to do a miniprep to purify our plasmid. We did PCR amplification of our gene fragments and purified our gene fragments to do Gibson assembly again. Small colonies grew on the plates with the yeast colonies, but we waited an extra day for more growth of colonies to do a yeast DNA extraction to do a gel visualization. Lastly, we ordered primers to verify the yeast assembly. Implementation: Our best results were from the CP-C vials with pH. However, they all lost the majority of the coagulation after being saturated by water for an extended period of time. Our rationale was that removing the supernatant would prevent this degradation of coagulation. We centrifuged and discarded the supernatant, leaving behind pancakes of pure white collagen.
September 27 - October 3: Gibson and Yeast Assemblies
As the last results of the Gibson Assembly were disappointing, we decided to redo a Gibson and a Yeast transformation using fresh stocks of reagents and the two-fragment and three-fragment assembly, which had the highest potential for successful transformation. Upon verification with PCR, our two-fragment Gibson Assembly was the only fragment that contained the correct length of overlap DNA, and thus was used in another Yeast Assembly and sent for sequencing.
October 4 - October 8: PCR and Yeast Analysis
We first started by checking our sequencing results from our Gibson assembly and picking colonies from the yeast plates to run PCR on overhangs to verify if the transformation worked. As the E. coli we believed that the assembly was successful, however the transformation was not successful. We used genomic yeast ethanol precipitation to extract the DNA for yeast assembly and LiOAc transformation. We performed PCR on _____, then prepared for a yeast miniprep and performed a gel using the pcr from the previous day. We extracted yeast cells from the plates that grew for 2 days following the transformation to do a yeast miniprep with in order to do a gel. Following our gel, we did an SDS page. We did a protein precipitation with zymolyase to break the yeast cell walls to do the SDS page. We streaked an E. coli plate to sequence with GIB for plasmid sequencing.
