Team:SDU-Denmark/Tour54
"In a dark place we find ourselves, and a little more knowledge lights our way." - George Lucas
Interlab study
Our team has been participating in the iGEM Interlab Study, to help further characterize three different promoters. This was done by using GFP-fluorescence as a way of measuring how strong each of the promoters are compared to each other.
The three constructs were made through iGEM standard biobrick assembly, inserting gfp into pSB1C3 plasmids containing the different promoters. The gfp-gene were originally cut out of pSB1A2, before ligation with each of the three promotes. Transformation of the constructs was done into E. coli K12 Top10, because it has been optimized for transformation and high levels of plasmid synthetisation.
Reference:One Shot® Top10 Chemically Competent E. coli. Catalog number: C4040-10. Visited: 17.09.15. [ThermoFisher]
According to iGEM headquarter protocol, biological triplets and technical triplicates was made for each promoter. Each construct has been sequenced and verified with colony PCR. For the complete protocol click here.
Each construct were sequences, two of the constructs were correct assembled, the third had the correct promoter, but showed no significant alignment to gfp.
For the measurements conducted in this project we used a FACSAriaII. The used method of the FACS were flow cytometry which functions through singling out droplets containing cells.
By exciting the proteins with light on the absorption frequency of the protein the cells will fluoresce and emission intensity can be measured.
We used E. coli K12 Top10 without inserted plasmids as our negative control and one of our own biobricks; pSB1C3-T18-LinkerGFP for the positive control. Top10 was used to calibrate the FACS and the arbitrary units (a.u.) was measured by gating.
Arbitrary units is relative to the measurements, it describes how strong one reading is compared to a defined reference.
Reference:Kate B. Bechtol, David K. Hanzel, and Bee
Choo Liang, Amersham Biosciences, Inc. - Understanding Fluorescence. [GelifeSciences]
The three promoters are part of a promoter family originally from a combinatorial library made by John Anderson in 2006. wt J23119 was made from synthetic oligonucleotides using overlap extension, and is the strongest of all the promoters in the familyREFERENCE WORD. Reference:iGEM Parts Registry - BBa_J23119:Design. Visited: 17.09.15. [Parts] J23101 is a constitutive promoter mutated form the wt J23119, and contains one point mutation in position -33, subsstituting G with T. J23106 and J23117 like J23101 are diverts of J23119, also constitutive promoters and modified in the same manner. J23106 have three mutations one in the -10 region and two in the -35 region, 8- and -9 plus -33 and -32 respectively. J23117 have two mutations in its -10 region, at position -8 and -12. All three promoters are 35 bp long. Reference:JCAnderson. iGEM Parts Registry - Visited: 17.09.15. [Parts] BBa_I13504 is a composite part composed of and RBS-site, a FACS optimized GFPmut3b and two standard biobrick terminators. The GFPmut3b has amino acid substitutions in position 65 and 72, a Glycine and Alanine respectively. Reference:Brendan P. Cormack, Raphael H. Valdiviaa, Stanley Falkow - FACS-optimized mutants of the green fluorescent protein (GFP). DOI:10.1016/0378-1119(95)00685-0. [ScienceDirect]
Top10 is the reference for the a.u. measurements compared to each of the other samples.
Our expectations of the promoters are listed from weakest to strongest in relative expression: J23117 < J23106 < J23101, based on the original measurements done in 2006.
In figure 1, a graph comparing each construct’s a.u. to the positive control, T18-Linker-GFP is depicted. The scale is logarithmic due to the relatively large difference in the measured expression levels of the promoters.
Sequence data for the three constructs was compared to the sequence retrieved from parts registry. It was correct for J23106-I13504 and J23117-I13504, but the data for J23101-I13504 did not show the gene encoding GFP, a BLAST search showed some alignment in the beginning of the sequence, to pFM46. Promoter J23106 showed the highest fluorescence of the constructs, with J23117 having a significantly (p < 0.0001) reduced fluorescence, at 40 times lower.
The J23106 construct showed a much higher fluorescence than the J23117 construct, with a mean value of 85932 a.u. compared to 814 a.u. see figure 2 for graph. The measurement on J23101 is inconclusive in this study, due to the wrongful sequence.