Week 1 (Aug 8):
As a part of our biotechnology pathway’s curriculum, we reviewed basic safety procedures and aseptic technique with our advisor Janet Standeven.
Week 2 (Aug 15):
Our focus this week was to ligate two of the three parts in our genetic construct: P-lambda-R--LacI--tsPurple--LAA/DAS and R0011--ClpXP--CI; however, we did not ligate the part without any degradation tag. After the ligations, the constructs were transformed into DH10 E. coli cells, but we were unsuccessful.
Week 3 (Aug 22):
Due to the unsuccessful transformations seen the previous week, we re-attempted our workflow (digest, ligation, transformation) of both parts. In addition, we ligated and transformed our third construct: P-lambda-R--LacI--tsPurple and R0011--ClpXP--CI.
Week 4 (Aug 29):
Transformations were ultimately unsuccessful because of RFP (red fluorescent protein) contamination. In addition, we miniprepped and nanodropped our genetic construct without the present degradation tag, which was in the 1C3 backbone.
Week 5 (Sep 5):
Due to several failures over the past few weeks, we attempted to detect previous errors by re-ligating and re-transforming the parts together using a different stock of P-lambda-R--LacI that was previously confirmed over the summer. Our results indicated faint, purple cells for our construct (P-lambda-R--LacI--tsPurple--R0011--ClpXP--CI), but no color was shown for the DAS and LAA cells after the transformation. In addition, we inoculated liquid cultures of our genetic constructs from the previous week’s transformations, including the genetic construct without the present degradation tag, the RFP colonies, and previous tsPurple colonies. Digests were performed of P-lambda-R--LacI--tsPurple--no degradation tag/DAS/LAA, R0011--ClpXP, and CI. After analyzing the gel, we concluded the tsPurple with no degradation tag/DAS/LAA, R0011--ClpXP, and CI were of expected sizes, but the P-lambda-R--LacI and R0011--ClpXP--CI digests were unsuccessful. Lastly, the following backbones were successfully digested for future ligations: 1A3, 1C3, 1K3, and 1T3.
Week 6 (Sep 12): Our constructs were sent out for sequencing; we found out that there was not an RBS between LacI and tsPurple. These results explained our transformation results from last week: while the cells would turn purple via read-through transcription, the cells with no degradation tag would become slightly purple, and the cells with degradation tags (DAS or LAA) would not have any color. At the same time of sending and receiving our sequences, we also hydrated B0034 from the iGEM kit of parts.
Week 7 (Sep 19): We had two sets of parts being constructed during this week. First, tsPurple with no degradation tag, DAS, and LAA were successfully digested and ligated into 1C3 backbones; after we transformed these vectors into NEB 10-beta cells. Second, we successfully miniprepped and nanodropped our B0034 part. After, we digested, ligated, and transformed B0034 and tsPurple with no degradation tag into 1C3.
Week 8 (Sep 26): Despite Lambert High School having fall break, our team was hard at work. We ligated and and transformed P-lambda-R--LacI--tsPurple--no degradation tag/DAS/LAA, but the cells did not turn purple as expected. Additionally, we went to Georgia Tech to practice presenting and to discuss our progress with Dr. Styczynski and Monica McNerney.
Week 9 (Oct 3): As a team, we decided to use premade GFP constructs that were built over the summer as our proof-of-concept. By doing so, we can verify results while simultaneously assembling our tsPurple constructs. After transforming our GFP constructs into DH10 cells, Keio Wild cells, and Keio ClpP Knockout cells, we sent the GFP constructs out for sequencing.
Week 10 (Oct 10): In the beginning of the week, we ligated P-lambda-R--LacI and B0034 together. We transformed three constructs: P-lambda-R--LacI--tsPurple--LAA, P-lambda-R--LacI biobrick in 3T5, and P-lambda-r--RFP in 3T5. Although we determined that our P-lambda-R--LacI--tsPurple--LAA construct was trash DNA, we were able to successfully obtain purple cells with the correct tsPurple construct P-lambda-R--LacI--tsPurple--no degradation tag/DAS/LAA--R0011--ClpXP--CI. As for our GFP constructs, we digested GFP with no degradation tag/DAS/LAA and ligated the parts in 1C3 backbone. These vectors were transformed into DH10 competent cells. Colonies of each construct in the three cell types were then inoculated into liquid culture in triplicates, and they were also induced with different levels of IPTG (no IPTG, 10uM IPTG, 100uM IPTG, and 1mM IPTG). Although they grew properly, we were unable to access a plate reader in time to analyze the levels of fluorescence from each tube. In addition, a series of transformations were done to verify that the constructs were properly made: P-lambda-R--LacI--GFP--DAS in 1AK3, P-lambda-R--LacI--GFP--DAS in 1C3, P-lambda-R--LacI--GFP--LAA in 1AK3, and P-lambda-R--LacI--GFP--LAA in 1C3. From the P-lambda-R--LacI--GFP--LAA in 1C3 construct, it was hard to identify colonies that had the correct green morphology, so a colony PCR was performed; we were able to verify that we had chosen the correct colony with GFP. From there, we inoculated the remaining colony into liquid culture and prepared for a miniprep.
Week 11 (Oct 17): We were able to successfully miniprep R0011--ClpXP--CI and P-lambda-R--LacI--GFP--ClpXP--CI, with final concentrations of 175.8 ng/uL and 79.1 ng/uL, respectively. Both constructs were sent for sequencing. In addition, we inoculated liquid cultures into 58 tubes of LB. We inoculated P-lambda-R--LacI--GFP, P-lambda-R--LacI--GFP--DAS, and P-lambda-R--LacI--GFP--LAA--ClpXP--CI into DH10, Keio Wild, and Keio ClpP Knockout cells in triplicates. In addition, we induced them into 0uM and 100uM IPTG levels. We also had four controls: a tube of plain LB, DH10 cells, Keio Wild cells, and Keio ClpP Knockout cells. Although the cells grew, none of the tubes showed fluorescence. As a result, we did another set of liquid cultures of P-lambda-R--LacI--GFP--no degradation tag/DAS/LAA--ClpXPCI in Keio Wild cells induced with 0uM, 10uM, and 100uM of IPTG into CArbenicillin LB. We also inoculated P-lambda-R--LacI--GFP--ClpXPCI in Keio Wild cells induced with 0uM, 10uM, and 100uM of IPTG into Tetracycline LB. Results TBD (Ms. Standeven is taking these down to the plate reader tonight).
Week 11 Miniprep R0011--ClpXP--CI and Week 11 Miniprep P-Lambda-R-LacI--GFP--ClpXP-CI