Part:BBa_K1678005:Experience
Characterization of the promoter functionality
This biobrick was assembled in pSB1C3 with the part BBa_K516030 that contains a RBS, the mRFP ORF and a double terminator.
For comparison, the biobrick BBa_J23119 was assembled with the same mRFP cassette on the same vector. These two plasmids were transformed in parallel in E. coli Top10 strain. The plasmid containing only BBa_J23119 without any coding sequence connected to it was use as a fluorescence-less control.
The cells were diluted to an OD600 of 0.01, grown to exponential phase and the fluorescence was measured on a TECAN plate reader when OD reached 0.3.
Excitation wavelength: 550 nm
Detection wavelength: 590 nm
The error bars represent the 95% confidence interval based on 12 independent measurements.
During the transcription, the RNA polymerase has to go through the LoxP array, which is made of repetitive sequences that are likely to form a hairpin. We show that this has an impact on the transcription efficiency (Mann-Whitney test, p-value < 10-6). However, it still allows for strong protein expression as the average expression level was equal to 91% of the expression level of the BBa_J23119 promoter.
Because brainbow-like systems usually require that only one copy of the sequence is present in the cell, we measured the expression level of the mCherry fluorescent protein bound to this promoter inside a chromosomally integrated cassette.
Excitation wavelength: 585 nm
Detection wavelength: 615 nm
The error bars represent the 95% confidence interval based on 12 independent measurements.
The average fluorescence is significantly different from the negative control (Mann-Whithney, p-value < 10-6).
Recombination by CRE recombinase
A cassette containing this promoter, the mCherry fluorescent protein (with RBS and terminator) followed by the for same LoxP sites in the same was inserted in the chromosome of E. coli. After plasmid expression of the CRE recombinase, the fluorescence disappeared, showing that recombineering can occur within these LoxP sites.
To the left: mCherry fluorescence imaged on colonies before expression of the CRE recombinase. To the right: mCherry fluorescence on colonies after the expression of the CRE recombinase.
More information can be found on http://2015.igem.org/Team:Paris_Bettencourt/Sustainability/Continuity
User Reviews
UNIQ7538333fe2aa0dd8-partinfo-00000004-QINU UNIQ7538333fe2aa0dd8-partinfo-00000005-QINU