Team:TU Delft/project/rbs characterization
From 2010.igem.org
RBS Characterization
For our RBS characterization project, five different RBS sequences from the Anderson RBS family (J61100, J61101, J61107, J61117, J61127) and the standard RBS B0032 were placed in front of the standard GFP coding sequence. The Biobricks generated in order to perform the experiments were: K398500, K398501, K398502, K398503, K398504. The general map of the construction is shown below, where the RBS is displayed in fucsia.
Feature | Function |
AmpR | Ampicillin resistance |
B0015 | Transcriptional (double) terminator |
B0062 | Transcriptional terminator |
E0040 | GFP |
G00000 | Standard prefix |
G00001 | Standard suffix |
G00100 | VF2 primer binding site |
G00101 | VR primer binding site |
J61100 | RBS Anderson family |
J23100 | Promoter |
The cells were cultured over 18 hours in 96-well plates using a Gen5 fluorescence and absorbance plate reader, LB with ampicillin was the culture medium used in these experiments. The protein production was measured as fluorescence production. The results are shown below.
RBS | Strength |
J61100 | 0.047513 |
J61101 | 0.119831 |
J61107 | 0.065454 |
J61117 | 0.038518 |
J61127 | 0.087334 |
B0032 | 0.300000 |
We've also looked at mRNA folded shapes using mfold to see if there was a common pattern in the Anderson RBS shapes. This might have been usable in predicting RBS strength for the other untested Anderson RBS sequences. Unfortunately, this didn't seem to be the case, as all the tried RBS sequences had very different mfold shapes.