Week 1
- Since the delivery of the parts and distribution kit took longer than expected we started working with plasmids that were included in the iGem Distribution Kit from 2021, PSB1A3 and PSB1C3 both with RFP construct.
- We grew a culture with E. coli DH5α containing Pcomb 3x given by PhD Alejandro Gonzales to make a MiniPrep and have a stock of the plasmid for further use.
- We prepared several stocks of chemocompetent cells and saved them at -80°C in aliquots of 200 μL for all the future transformations procedures.
Figure 1. Performing a miniprep of PSB1A3 and PSB1C3 from the iGem Distribution Kit 2021
Week 2
- We transformed PSB1C3 and pBLUESCRIPT into DH10B and DH5α strains, respectively, to test the transformation efficiency of each cells.
- We grew a colony of the transformed pBLUESCRIPT given by CEM team and made a MiniPrep to save a stock of the plasmid and prepared a stock of the culture to be conserved at -80 °C in LB media with 13% glycerol.
- We transformed PSB1C3 and PSB1A3 into E. coli DH5α cells and obtained a few colonies and picked two of those colonies from each plasmid to miniprep, also we induced both constructs with IPTG to confirm functionality of the RFP construct and made stocks at -80 °C in LB media with 13% glycerol.
- An experiment to determine the optimal digestion time was performed with the enzymes PstI and EcoRI. In each sample we only changed the time using 1, 2 and 3 hours. The digestion products were run on an 1% agarose gel and the optimal time was 3 hours since this time gave us better band definition and less degraded DNA.
Figure 2. Expression of RFP in the E. coli DH5α after the induction with IPTG
Week 3
- Cultures of NEB 10 competent cells were grown overnight to propagate the strain and create new stocks of calcium competent cells.
- A culture of TG1 strain was also grown overnight to save an aliquot at -80°C.
- Digestions of PCB1C3 with the enzymes PstI and EcoRI were performed in order to test the performance of our enzymes against a stock provided by the academic department. It was determined that the enzymes owned by the team had higher efficiency with a 2 hour digestion and an enzyme inactivation at 80°C for 20 min. This did not explain the low quantity of digested plasmid product.
- Recovery of the digested plasmid from the agarose gel after electrophoresis was done using the Monarch gel purification kit, low concentrations of the plasmid DNA was recovered and quantified in the nanodrop.
Figure 3. Agarose gel 1% used to analyse the digestion products. From left to right: ladder of 1 kb, negative control, 1 hour digestion, 2 hours digestion, 3 hours digestion. All the digestions show degraded DNA and the 3 hour digestion was determined to be the optimal due to a better band definition and less degraded DNA.
Week 4
- The first attempt of VCSM13 bacteriophage propagation was performed by infecting a 2mL TG1 culture using 2 μL of bacteriophage stock and incubating for 1 hour at 37°C and 250 rpm.
- The 2 mL of the infected culture were transferred to 2 falcon tubes with 15 mL of LB media each and incubated for 1 hour at 37°C and then kanamycin was added to both and incubated at 37°C overnight.
- The recovery of the M13 bacteriophages was performed by centrifugation and the supernatant was saved as stock.
Week 5
- The parts we synthesized with IDT arrived and after resuspension we made a PCR to verify its integrity. The PCR product was run on a 1% agarose gel.
- We started to prepare all the necessary for the cloning of the gBlocks. The digestion of the backbones was performed and we noticed big amounts of degraded DNA were in the bottom of every lane of the electrophoresis gel. At this point we were obtaining high recovery yields from the MiniPreps (2000-5000 ng/μL), so we expected a good amount of digested backbones.
- To solve this problem we performed each digestion by triplicate to concentrate and get a higher amount of digested backbone, however concentrations were low, less than 10 ng/ul.
Figure 4. Agarose gel 1% used to analyse the digestion products. From left to right: ladder of 1 kb, PSB1A3 by triplicate, PSB1C3 by triplicate, Pcomb3x by triplicate. Note the degraded DNA in each digestion reaction
PCRs at different annealing temperatures (a range of 55°C to 70°C) to identify the optimum temperature of Axtl primers were performed. 61°C was the best annealing temperature for this set of primers
Week 6
- We digested the parts and proceeded with a fast ligation (10 min at room temperature) for the first cloning attempt with the IDT gBlocks.
- We transformed the ligations and had not successful results.
- Twist parts arrived, after they were held by the custom house for two months at room temperature. We amplified the gBlocks by the first time and identified that the parts presented considerable degradation, also we could not amplify from PCR products, indicating poor DNA integrity with the parts.
Figure 5. Agarose gel 1% showing the degradation of the Twist parts
Week 7
- To avoid the degraded plasmidic DNA, and try to have more digested plasmids, we changed the minipreps solutions with freshly made ones and also tested other ones borrowed to make a comparison between the quality of the recovered DNA.
- Even with this strategy we still presented a lot of degraded DNA so we decided to use a borrowed kit to make the MiniPreps, we obtained a lower recovery yield (250-450 ng/μL), but with a better quality, since no degraded DNA was identified by 1% electrophoresis gel.
- Digestions with the kit minipreps were performed and we obtained acceptable amounts that we could recover from the gel (9-34ng/μL), for this we concentrated with magnetic beads a single miniprep of each plasmid in a lower volume.
Figure 6. Agarose gel 1% used to analyse the digestion products. From left to right: ladder of 1 kb, PSB1A3 by duplicate using 15 enzyme units, PSB1C3 by duplicate using 15 enzyme units, Pcomb3x by duplicate using 15 enzyme units, PSB1A3 concentrated, PSB1C3 concentrated, Pcomb3X concentrated. Note the supperior yield and lack of inespecific activity on the last 3 reaction
Week 8
- With the new digested backbones using MiniPreps from kit, the second clonation attempt was done using fast ligation (10 min at room temperature), and a high molar ratio of approximately 5:1 insert-backbone.
- The third clonation attempt took place, with new digested parts and using longer ligation times (16°C overnight) and a higher molar ratio approximately 7:1 insert-backbone. Two ligation products provided colonies and were tested, giving a positive colony PCR confirming the insert was successfully cloned. The need for higher insert:vector yield could be explained by the unavility of the parts to be accessible for ligations as consequence of the degradation process
- Digestions with the kit minipreps were performed and we obtained acceptable amounts that we could recover from the gel (9-34ng/μL), for this we concentrated with magnetic beads a single miniprep of each plasmid in a lower volume.
- Digestions with the kit minipreps were performed and we obtained acceptable amounts that we could recover from the gel (9-34ng/μL), for this we concentrated with magnetic beads a single miniprep of each plasmid in a lower volume.
Figure 7. Agarose gel 1% used to analyse the colony PCR products. From left to right: Control, Colony 1 PSB1A3+FinalCamRNA, Colony 2 PSB1A3+FinalCamRNA, Colony 1 PSB1A3+ManualCamRNA , Colony 2 PSB1A3+ManualCamRNA