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Team:Groningen/Template/MODULE/Notebook/detection
From 2014.igem.org
Notebook
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Detection
July 28 - August 3
The following BioBricks were taken from the 2014 iGEM distribution kit, and transformed to E. coli DH5α
The double terminator(BBa_ B0015)
The RBS(BBa_ B0034)
The gene for the purple-blue chromoprotein amilCP(BBa_ K592025)
The genes for the AIP sensor infrastructure(BBa_ I746101)
The gene coding for GFP(BBa_ E0040)
The lasR gene(BBa_ S03156)
The promoter LasR(BBa_ R0079)
The lasI protein generator(BBa_ K081009)
All samples were digested, and checked on gel
From all transformations a glycerol stock was made, and stored at - 80 °C
August 4 - August 10
A new batch of cmpetent E. coli DH5α was prepared
A ligation was performed with the purple-blue chromoprotein amilCP BioBrick (BBa_K592025) together with the double terminator BioBrick (BBa_ B0015) with the pSB2K3 vector
The ligation product was transformed to E. coli DH5α and plated on LB agar with kanamycin
An overnight culture was grown, and the plasmid was isolated out of the culture in order obtain a high concentration of construct
The construct was double digested, and checked on gel
August 11 - August 16
Two ligation were performed, the first one was the purple-blue chromoprotein amilCP with the double terminator was ligated with promoter CP44 (BBa_K1033225), the second one was the purple-blue chromoprotein amilCP with the double terminator was ligated with promoter CP29 (BBa_K1033222)
Both constructs were transformed to E. coli DH5α
Some blue colonies were visible on the plates, an overnight culture was prepared from these colonies
The plasmids were isolated out of the overnight cultures
The size of the constructs were digested, and checked on gel
August 24 - August 31
Nine DH5α strain containing each a different insert were inoculated from the glycerol stock, resulting in a culture containing the following parts: the double terminator (BBa_ B0015), the RBS, the genes for the AIP sensor infrastructure (BBa_ I746101), the gene coding for GFP (BBa_ E0040), the promoter CP29 (BBa_K1033222), the lasR gene (BBa_ S03156), the lasI protein generator (BBa_ K081009), pSB1C3 with mrfp pSB1C3), and pSB1A3 with mrfp pSB1A3)
The plasmids containing the parts were isolated out of the cultures, and the mentioned part were digested and purified out of the plasmid.
Three ligation were perfomed, the lasR with the double terminator, the gene coding for GFP with the double terminator, and lasI protein generator
All ligation mixtures were transformed to E. coli DH5α
September 1 - September 7
The size of the construct was determined by colony PCR
The cultures, which showed positive results for the colony PCR were inoculated in fresh LB media containing the corresponding antibiotics
Two new ligations were performed, the lasR double terminator was ligated with Promoter CP29, and the gene coding for GFP double terminator was ligated with the RBS
The two new construct were both transformed to two different batches of E. coli DH5α
The size of the construct was checked by colony PCR
September 8 - September 14
All construct were isolated out of E. coli DH5α, and send for sequencing
September 15 - September 21
The following parts were confirmed by sequencing
The AHL generator in vector pSB1A3 (BBa_ K1365998)
GFP with RBF and double terminator in vector pSB2K3(BBa_ K1365999)
The vectors pIL253 is send to the registry and checked on gel (BBa_K1365301)
We also tried to BioBrick the pNZ8048 vector
September 22 - September 27
Growing Lactis in a media containing heme, in order to collect some data for the modeling
Performing nisin activity tests with L. lactis strain NZ9700, for different glucose concentrations
September 28 - October 4
Samples were sent for sequencing
The sequence of the AHL sensor was confirmed in pSB1A3 (BBa_K1365997)
October 5 - October 10
The following parts were successfully constructed in pSB1C3, and sent to the registry
The AHL generator (BBa_ K1365998)
GFP with RBF and double terminator (BBa_ K1365999)
The AHL sensor (BBa_K1365997)
