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Team:Groningen:Notebook:Characterisation
From 2014.igem.org
Notebook
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Characterization
September 1 -September 7
Performing a positive control for the NisA contruct (BBa_K1365556)
Plated L. lactis strain NZ9000 cells on the M17 agar plates
Made pores on the plate and added L. lactis strain NZ9700 strain in the well
Overnight incubation in 30 °C incubator
Zone of inhibition was observed.
September 8 - September 14
Obtaining the NisA (BBa_K1365000) and the Superfolded GFP (BBa_K1365020) BioBricks in larger concentration for further use
Transformed both, pSB1C3 containing the NisA BioBrick and pSB1C3 with the Superfolded GFP Biobrick, to two different E. coli DH5α strains
The transformations were checked on gel, and the desired bands were observed, therefore the transformation was most likely successful
The plasmids with the desired BioBricks were isolated out of E. coli DH5α, and glycerol stocks were made of the two strains containing the previous named parts
September 15 - September 21
Fusion of the NisA BioBrick (BBa_K1365000) and the Superfolded GFP BioBrick (BBa_K1365020) with a double terminator (BBa_B0015)
The size of the plasmids were confirmed by single and double digestion of the plasmids (pSB1C3) with the NisA and Superfolded GFP BioBricks to confirm the sizes of the plasmids
Both, the NisA and the Superfolded GFP BioBricks, were ligated into pSB1A3 together with the double terminator as a second insert
The ligation was performed overnight, after which the ligated plasmids were transformed to two different E. coli DH5α strains
The size of the construct was determined by colony PCR
A double digestion was performed, and the constructs were further purified
September 22 - September 28
Fusion of the NisA with the double terminator and Superfolded GFP with the terminator constructs with the RBS BioBrick (BBa_B0034) and purification of the promoter collection of Uppsala containing the following promoters: CP1 (BBa_K1033219), CP11 (BBa_K1033220), CP29 (BBa_K1033221), CP30 (BBa_K1033223), CP41 (BBa_K1033224) and CP44 (BBa_K1033225)
Both, the NisA with the double terminator and Superfolded GFP with the terminator constructs, were ligated into pSB1K3 together with the RBS BioBrick as a second insert
The ligation was performed overnight, after which the ligated plasmids were transformed to two different E. coli DH5α strains
The size of the construct was determined by colony PCR
Several positive clones were inoculated, and their plasmids were isolated after inoculation
One of the most important and basic requirement for a gold medal is to improve the function OR characterization of an existing Biobrick part. So we decided to characterize the Biobricks designed by our own team.
September 29 - October 5
Testing the promoter collection containing CP1 (BBa_K1033219), CP11 (BBa_K1033220), CP29 (BBa_K1033221), CP30 (BBa_K1033223), CP41 (BBa_K1033224) and CP44 (BBa_K1033225), and testing the newly formed RBS-NisA-double terminator (BBa_K1365556) and RBS-Superfolded GFP-double terminator (BBa_K1365555) BioBricks
The RBS-NisA-double terminator BioBrick was ligated with CP29 and CP44 from the promoter collection
The RBS-Superfolded GFP-double terminator BioBricks was ligated with all the promoters of the promoter collection
October 6 - October 12
Testing the constructs RBS-NisA-double terminator (BBa_K1365556) and RBS-Superfolded GFP-double terminator (BBa_K1365555) with some different promoter: P2, PLas, and two constitutive promoters (BBa_J23101 and BBa_J23115), and inserting these constructs in pIL253 (BBa_K1365301) vector
The ligations of the different constructs were performed overnight, after which the ligated plasmids were transformed to L. lactis NZ9800, and finally a colony PCR was performed with several colonies of each construct
The construct with the P2 and PLas did not show any bands on the gel, therefore further steps were only done with the constructs containing the two constitutive promoters
The same constructs with the promoters from the promoter collection of Uppsala (see September 19 till October 5) were subjected to ligation again, but this time the vector pIL235A was used instead of pSB1K3
These constructs were transformed to L. lactis NZ9800, and plated on M17 agar with erythromycin
October 13 - October 17
Characterization of the BioBricks with the help of FACS
The colonies, which grew on the plates were subjected to FACS, but unfortunately no fluorescence was observed in the L. lactis
The same constructs were also tested in E. coli, which showed fluorescence
