Team:LambertGA/Notebook
Notebook
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.
Our focus this week was to ligate two of the three parts in our genetic construct: P-lambda-R--LacI--TS Purple--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.
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--TS Purple and R0011--ClpXP--CI.
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--TS Purple--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 TS Purple colonies. Digests were performed of P-lambda-R--LacI--TS Purple--no degradation tag/DAS/LAA, R0011--ClpXP, and CI. After analyzing the gel, we concluded the TS Purple 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): We sent out constructs for sequencing and found that our constructs did not have an RBS between LacI and Tspurple, so our cells would turn purple via read-through transcription, meaning only cells with TsPurple (no deg Tag) would be slightly purple but a deg tagged TsPurple would result in no color at all.
B0034 was hydrated from iGEM kit of parts
Week 7 (Sep 19): Digestion of TsPurple (no deg tag/DAS/LAA) and ligation into 1C3 vector. Then we transformed into NEB 10-beta cells
Digestion of B0034 and TsPurple (no deg tag/DAS/LAA)
Results: all successful
Ligation of B0034 & TsPurple (no deg tag/DAS/LAA)
Transformation of B0034 and TsPurple (no tag/DAS/LAA)
Results: Successful growth
Week 8 (Sep 26): Fall Break
Week 9 (Oct 3): Decided to use premade GFP constructs as a proof-of-concept while simultaneously building our Tspurple constructs
Transformed GFP constructs into three types of cells: DH10, Keio Wild, and Keio ClpP Knockout
Results: Mostly Unsuccessful. Many of the Transformations were effective, but the construct was not functioning as it should have been
GFP Constructs sent for sequencing to determine if there is an error in the construct
Week 10 (Oct 10):
Ligation of P-lambda-R-LacI and B0034
Digestion of P-lambda-R--LacI--GFP (no tag/ DAS/LAA) and ligation into 1C3 Backbone. We then transformed into DH10 competent cells
Liquid cultures of each GFP construct in each cell was made with varying levels of IPTG; No IPTG, 10 uM, 100 uM, 1 mM, in triplicate
Transformation of:
- P-lambda-R--LacI--GFP (DAS) [from past miniprep] in 1AK3
- P-lambda-R--LacI--GFP (DAS) [from ligation] in 1C3
- P-lambda-R--LacI--GFP (LAA) [from past miniprep] in 1AK3
- P-lambda-R--LacI--GFP (LAA) [from ligation] in 1C3
- P-lambda-R--LacI--TsPurple (LAA) [Curious if it worked] in 1AK3
- P-lambda-R--LacI Biobrick in 3T5
- P-lambda-R--RFP in 3T5
- R0011--ClpXP--CI Biobrick in 1AK3
All successful with an exception of P-lambda-R--LacI--TsPurple (LAA), which concluded that it was trash DNA and was thrown out. P-lambda-R--LacI--GFP (no tag/DAS) [from ligation] in 1C3 were innoculated into liquid cultures to miniprep. Morphology was difficult to determine in P-lambda-R--LacI--GFP (LAA) in 1C3 as the GFP was very hard to detect. To make sure we picked a colony that had the insert, we performed a colony PCR
Results: Colony picked had correct DNA insert. Inoculated liquid culture from colony to miniprep.
Purple cells were finally achieved due to successful TsPurple constructs of: P-lambda-R--LacI--TsPurple(no tag/DAS/LAA)--R0011--ClpXP--CI
Week 11 (Oct 17):
Successful miniprep of R0011--ClpXP--CI and P-lambda-R--LacI--GFP--ClpXP--CI
R0011--ClpXP--CI had a final concentration of 175.8 ng/uL P-lambda-R--LacI--GFP--ClpXP--CI had a final concentration of 79.1 ng/uL
Both constructs were sent for sequencing
Inoculation of 58 tubes in Plain LB
- P-lambda-R--LacI--GFP into DH10, Keio Wild, and Keio ClpP cells as well as induced into 0 uM and 100 uM IPTG levels in triplicates (18)
- P-lambda-R--LacI--GFP (DAS) into DH10, Keio Wild, and Keio ClpP cells as well as induced into 0 uM and 100 uM IPTG levels in triplicates (18)
- P-lambda-R--LacI--GFP (LAA)--ClpXP--CI into DH10, Keio Wild, and Keio ClpP cells as well as induced into 0 uM and 100 uM IPTG levels in triplicates (18)
- Four controls: plain LB, DH10 cells, Keio Wild type cells, and Keio ClpP cells
