Team:LambertGA/Notebook
Notebook
Safety procedures and aseptic technique was reviewed by our iGEM supervisor: Janet Standeven.
This week was subjected to ligating two of our three parts of the genetic construct together: p-lambda-r/Lac I/ tspurple/ deg. Tag (LAA or DAS tags) + ROO1/ ClpXP/ CI
After ligation was complete the the genetic constructs were then transformed into separate DH10 E. coli cells Results: Unsuccessful for unknown reasons. Looking back it was most likely a procedure-error or a lack of a B0034 RBS binding site
The previous transformation and ligation was unsuccessful due to the lack of ribosomal binding sites in the genetic constructs
We were required to re-digest, re-transform and re-ligate the p-lambda-r/ LacI/ TsPurple/ deg.tag(LAA or DAS) + ROO11/ ClpXP/ CI
Transformation and ligation of the third part: genetic construct without a degradation tag present
The continuation of the re-ligations and re-transformations during week 3 were ultimately unsuccessful due to RFP(red fluorescent protein) contamination and/or lack of functioning genetic conscruct
Minipreped and nanodroped the genetic construct without a present degradation tag in the backbone 1C3.
Further detection of previous errors
Liquid culture of genetic construct without degradation tag
Inoculation of RFP and previous TS purple colonies
Performed digests of p-lambda-r/LacI, Ts purple (no deg tag/ DAS/ LAA), R0011-ClpXP-CI, R0011-ClpXP, and CI.
Show picture of gel
Conclusion: TsPurple(no deg tag/ DAS/LAA), R0011-ClpXP, and CI digests were of expected sizes and p-lambda-r-LacI, and R0011-ClpXP-CI digests were unsuccessful
Digested 1A3, 1C3, 1K3, and 1T3 backbones for future ligations
Show gel
Conclusion: all successful
Due to several unsuccessful procedures, we re-ligated p-lamba-r/LacI + Ts purple (no deg tag/DAS/LAA) and R0011/ClpXP + CI using a different stock of p-lambda-r-LacI that was previously confirmed
The above ligations were also transformed
Results: very faint cells for p-lamba-r/LacI + Ts purple (no deg tag) and no color for DAS or LAA cells
Liquid culture of genetic construct without degradation tag
Inoculation of RFP and previous TS purple colonies
Performed digests of p-lambda-r/LacI, Ts purple (no deg tag/ DAS/ LAA), R0011-ClpXP-CI, R0011-ClpXP, and CI.
Show picture of gel
Conclusion: TsPurple(no deg tag/ DAS/LAA), R0011-ClpXP, and CI digests were of expected sizes and p-lambda-r-LacI, and R0011-ClpXP-CI digests were unsuccessful
Digested 1A3, 1C3, 1K3, and 1T3 backbones for future ligations
Show gel
Conclusion: all successful
Due to several unsuccessful procedures, we re-ligated p-lamba-r/LacI + Ts purple (no deg tag/DAS/LAA) and R0011/ClpXP + CI using a different stock of p-lambda-r-LacI that was previously confirmed
The above ligations were also transformed
Results: very faint cells for p-lamba-r/LacI + Ts purple (no deg tag) and no color for DAS or LAA cells
Successful transformation of TsPurple and GFP constructs with variation of degradations tag (LAA, DAS and no degradation tag) and IPTG levels ( 1 uM, 10 uM, 100 Um, 1 mM)
Ligation
