Today was our first day back at the lab starting this new fall semester! Most of our usual lab group was not able to attend, so we spent time discussing how to move forward and how our new lab schedule was going to work with classes beginning. We also made stocks and created more plates with the necessary antibiotics. We created three batches of agarose to make agar plates containing Kanamycin, Chloramphenicol and plates with both antibiotics. It was the first time making plates for some of the lab group, and they experienced quite a learning curve regarding how quickly the agarose gel would set as it cooled. Overall, it was a good learning experience!
On this day of lab, we focused on creating gels to be able to run the Gibson Assembly and other processes performed last week. Three different gels were created using the standard protocol. After waiting a considerable amount of time, the gels had still not set, which indicated something had gone wrong. It was then discovered that we had used the incorrect powder- as instead of using agarose powder or LB agarose powder, we had used an LB Agar powder. Unfortunately, we did not have time to remake the gels before our time in the lab was over, but we had utilized the time we spent waiting working on other aspects of wet lab project- including discussing write-ups, important concepts, and deadlines. Even though we all have spent a considerable amount of time in the lab, we were reminded that mistakes can still happen, and there is always more to learn!
For the first day in lab this week, we were working on making several aliquots of Kanamycin and Chloramphenicol. We made 1.5mL Kanamycin aliquots by adding 300mg Kanamycin and 10mL of DI water, vortexing and filtering the solution with a syringe into the seven tubes! We repeated the process to make 1.0mL of the Chloramphenicol aliquots, but instead added 250mg and 10mL of 99.5% ethanol solution. These were stored in the –80C freezer. We concurrently made agarose gels to run gel to test our previous Gibson assembly. Lastly, one of our lab members picked colonies from Linalool plates and double transformed pJBEI_6409 to make several overnight cultures.
For lab today, we made four 1mL aliquots of the linalool double transformation overnight cultures from the day before. At the same time, other members worked on back dilutions to induce the linalool. We added different concentrations of IPTG into the following conditions to test for which one would result in the best expression!
For our last day in lab this week we performed a cell viability assay of the Linalool samples! We also plasmid prepped colony 18 by following the Monarch Plasmid miniprep kit protocols. Then we nanodropped our results and fortunately we had a high concentration of 53.2ng/ul of Menthol! Exciting! We then sent the samples for sequencing. We then performed two batches of PCR on the Linalool_vector_1. To do this we reconstituted the primers spinning them down and resuspending them in water. Then we made aliquots of the primer followed the protocol for PCR. At the end of lab, we collected samples of Linalool double transformations overnights and followed the protocol.
Unfortunately, despite our excitement over the nanodrop results showing 53.2 ng/µL of menthol, which was a high enough concentration for us the proceed with, the sequencing came back unsuccessful, which means we can no longer move forward with menthol. This was a bit of a disappointment for the team. However, there’s some good news! Our work with linalool was successful. Today, we prepared three gels and ran a gel electrophoresis on the linalool PCR reaction from last week, expecting a band around 6,800 bp. The gels looked great with bands at exactly about 6800 bp, and we were able to move forward with PCR purification. We spotted the four induction conditions and four different cultures on a plate. We also set up overnight cultures of three working clones of iGEM_003 in LB with Cam25 and Kan30.
The team had a busy day today! We back-diluted and induced the iGEM_003 overnight cultures, incubating them at 30°C. We ran four induction conditions across four different cultures to cover all our bases. We also completed a Gibson Assembly on the PCR product, transforming it into competent DH5α cells, and plated them on Kan30 plates at 1:10 dilutions. We took samples from yesterday’s overnights to run another cell viability test, making sure everything is on track. Lastly, since we ran out of Kan plates, we made more plates to keep the workflow running smoothly. The team is making steady progress, despite a few setbacks.
Today marked our last day in the lab space we’ve been using since the summer. The team worked hard to wrap up any remaining experiments that needed to be conducted in the lab. We ran colony PCR on eight colonies from the Gibson Assembly completed yesterday. Additionally, we assessed cell viability on the overnight cultures once again. Half of the team focused on packing up equipment and materials for transfer to another lab for HPLC analysis. All linalool samples will be run on HPLC to test for results next week in professor Woolston’s lab.