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

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

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

Fall Break (We did stuff here)

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

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

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