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 |
RBS | Strength |
J61100 | 0.047513 |
J61101 | 0.119831 |
J61107 | 0.065454 |
J61117 | 0.038518 |
J61127 | 0.087334 |
B0032 | 0.300000 |
The RBS strength was calculated by taking the mean of the ratio between the expression of the standard RBS (B0032) and expression of Anderson RBS over time. Expression being the measured fluorescence divided by measured biomass (absorbance, OD).
The measurements were taken from the part of the curve where optimal growth can be assumed; which occurred from 0:40 until 2:30, approximatively. The result is a simple characterization of the Anderson RBS sequences in relation to the known RBSes. The given strengths are displayed taking the standard RBS B0034 as reference.
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.