For week 5 we used the nanodrop to measure the concentrations of the PCR purified samples from the prior week. Exciting! These samples included reactions 1 and 3 for the pJBEI plasmids, and both the Menthol and Linalool reactions for PET28a. Unfortunately, since our pJBEI reaction 2 didn’t work after running the gel last week, no bands were visible, we did not check the concentrations for this reaction. The following concentrations were: pJBEI Rxn 1 Sample 1: 40.8 ng/uL pJBEI Rxn 1 Sample 2: 58.5 ng/uL pJBEI Rxn 3 Sample 1: 41.8 ng/uL pJBEI Rxn 3 Sample 2: 32.0 ng/uL PET28a Menthol: 54.4 ng/uL PET28a Menthol Rxn 2 : missing PET28a Linalool Rxn 1: 26.0 ng/uL PET28a Linalool Rxn 2: 29.3 ng/uL Once the concentrations of the working reactions were obtained, all lab members learned about Gibson assembly. Then we started working on the Gibson Assembly for Menthol and Linalool! Based off the concentrations we collected, our team calculated the amounts required to carry out the Gibson assembly for Menthol and Linalool! For the Gibson assembly of Menthol we used 1.25 uL for each of the three Menthol fragrances. For the Gibson Assembly of Linalool we used 3uL of one Linalool fragrance! Once we finished this, we did PCR on both Gibson Assemblies and a colony PCR for reaction 2 pJBEI!
For week 6 we started by re-running a gel on pJBEI-6409 reaction 2, since it failed when running the gel on it last week. To prep, four lab members made the gel using 1X TAE buffer, Syber safe, and agarose! Once the gel solidified, we prepped the PCR results with 1uL of 6X stain and ran the gel at 120 Volts. To save more time and get our gel results sooner, we imaged our gel after letting it run for 20 minutes! After analyzing the results, we learned that our gel failed again for reaction 2. On the bright side, our team discussed possible errors for why our PCR failed, such as primer design error, incorrect time, temperature errors in transformation, or the cells don’t have the plasmid! Considering these possible errors, we decided on a new course of action to potentially create a successful reaction 2. We considered using liquid culture, moving forward, and we decided to test if the antibiotic was present on the plates to know if we were using the right plasmid! For our Gibson Assemblies we worked on last week, we began transforming them into DH5alpha by adding 2uL of competent cells and following the protocol. Once we finished the protocol, we incubated the result at 37C overnight. While part of the team was working on this transformation, the rest of the team made four overnight stocks of the pJBEI by picking colonies from the 200ul pJBEI plate to grow them at 37C. We also made more Kanamycin resistant plates to select the transformed cells! Despite the downfalls in today’s lab, we were very productive, for we made progress with our Gibson assemblies and gained a better understanding of what to do going forward!
For the second day of lab, we started off by reconstituting primers at 6800g for 3 mins. Then in separate tubes we carried out a 10x dilution to the primers 13 to 18, which contained the Menthol’s, Linalool’s, and pBEI’s forward and reverse primers. After following the protocol for the 10x dilution, we put the original primers back into the freezer to keep the stock safe. After this, we started a colony PCR on the Menthol and Linalool. We first prepped the DNA templates by taking 6 circled colonies from Menthol and from Linalool and put them in their corresponding labeled tubes with 100ul of nuclease free water! Using these DNA templates, DI water, One Taq buffer, and the diluted primers prepped earlier today, we assembled and ran a colony PCR on the Menthol and Linalool Gibson Assembly! Though we couldn’t finish our PCR in time to run gels on them, we decided to push this to the third day of lab this week. Towards the end of lab, we nano-dropped the concentrations for our pJBEI which showed: PJBEI-6409 tube 1A: 0.0117 ug/uL=11.7ng/uL NEW 14.1ng/uL PJBEI-6409 tube 1B: 0.0138 ug/uL =13.8ng/uL NEW 15.0 ng/uL PJBEI-6409 tube 2A: 0.0285 ug/uL = 28.5ng/uL PJBEI-6409 tube 2B: 0.0111 ug/uL= 11.1ng/uL PJBEI-6409 tube 3A: 0.0125 ug/uL (nothing in tube)= 12.5ng/uL NEW 11 ng/uL PJBEI-6409 tube 3B: 0.0092 ug/uL= 9.2 ug/uL NEW 11.8ng/uL PJBEI-6409 tube 4A: 0.0228 ug/uL= 22.8ng/uL PJBEI-6409 tube 4B: 0.010 ug/uL = 1.0ng/uL Outside of lab, two other team members did an overnight culture by combining 400uL of culture into 5000uL of LB and induced the culture with IPTG and an antibiotic.
For the third day of week 6, we started where we left off running the gel on the colony PCR of our Gibson assemblies for Linalool and Menthol! We all loaded 10uL of the PCR results into the 12 wells and decided to add the DNA ladder in the well between the 6 menthol wells and 6 Linalool wells to make it easier to compare and analyze the bands! Unfortunately, all the Menthol 5800 loaded wells did not work however, the Linalool 1850 worked for two columns! We planned to rerun a colony PCR for the menthol in the following week! Aside from running the gels, we also plasmid miniprepped Menthol 1 through 6, and Linalool 1 through 6 by following the plasmid purification protocol. To wrap up our lab, we collected samples by aliquoting 1mL of each of the four liquid cultures and spinning them in the microcentrifuge to separate the supernatant from the pellet. When we measured the OD using the nanodrop the results of the four samples were the following: 2.68 ng/uL 2.84 ng/uL 2.81 ng/uL 2.71 ng/uL
On the first day of week 7, we purified pJBEI6409 and reran the PCR for all 6 menthol colonies that were picked from last week. Additionally, we ran the nanodrop for menthol Gblocks and the concentrations showed:
These concentrations are decent for us to proceed with our next steps!
Great news today-transformation for 2 of the plates was successful! A promising amount of isolated single colonies showed on the 200uL pure menthol plate whereas the 100uL pure menthol plate showed fewer colonies. The team decided to pick 6 colonies from the 200uL plate and 2 colonies from the other plate. While that was happening, other team members ran a gel to check the linalool PCR, and the linalool samples looked good to send for sequencing. With the remaining time, we made TSS Buffer to prepare for making competent cells next week!
Today, we ran a gel on the menthol colony PCR to verify successful assembly (Results will be checked tomorrow). We also prepared 12 Kanamycin plates by adding 8g of LB Agar to 200 mL of DI water and incorporating 150 mg of Kanamycin sulfate, which was sterilized using a vacuum filter. These plates were used to make the liquid overnight cultures of BL21DE3_pJBEI6409, which were prepared by inoculating 3 mL of LB media with Cam25 and a sterile ice stab from the -80°C freezer. We then moved on to redoing the menthol Gibson Assembly using the pET28a vector due to the first assembly being unsuccessful. This involved mixing 2 μL of each menthol fragment and 1 μL of the pET28a vector with 5 μL of Gibson Master Mix (another batch was made by mixing 1 μL of each menthol fragment and 2 μL of the pET28a vector with 5 μL of Gibson Master Mix to see if the fragment to pET28a ratio was a factor in the unsuccessful Gibson Assembly). After assembling the DNA fragments, the mixture was incubated at 50°C for one hour in a thermocycler. Once completed, we transformed 2 μL of the Gibson reaction into competent DH5α cells from the NEB Gibson Assembly kit.
Today, we focused on preparing competent BL21DE3_pJBEI6409 cells, which involved inoculating 25 mL of LB media with Cam25 and 500 μL of overnight culture. After growing the culture to an OD between 0.3-0.5, we transferred the cells to a chilled 50 mL tube and placed it on ice for 20 minutes before centrifuging. The cell pellets were then resuspended in TSS buffer, and 50 μL aliquots were stored at -80°C. Later in the day, we performed colony PCR on the Gibson Assembly from Monday, picking 40 colonies (20 colonies from the 2-2-2-1 and 20 from the 1-1-1-2) and inoculating them in water. Unfortunately, while adding the One Taq, FWD menthol primer, REV, menthol primer, and DI water into the 40 tubes of each template, we ran out of Taq! So, for now, we ran the thermocycler using the PCR protocol for the tubes that were filled and are going to wait to do the next few tubes tomorrow when we receive more Taq. Finally, we set up a PCR reaction using the pET28a vector for menthol. The thermocycler protocol included an initial denaturation at 98°C for 30 seconds, followed by 30 cycles of denaturation, annealing, and extension at 98°C, 56-58°C, and 72°C, respectively. A DPN1 digest was performed to remove methylated template plasmids, which were then stored at -20°C for purification the next day.
Today, we focused on transforming the PET28a_Linalool plasmid into the newly prepared competent BL21DE3_pJBEI6409 cells. We also conducted a control transformation of the empty pET28a vector. Both transformations followed standard heat shock and SOC media incubation protocols. After spreading the cultures on Kanamycin plates, they were incubated overnight at 37°C in the Woolston lab for further analysis and future experiments. Unfortunately, we did not get more Taq today, so we will have to wait until next week to perform the rest of the colony PCRs.