Team:TU Delft/project/rbs characterization

From 2010.igem.org

Revision as of 13:57, 10 August 2010 by Hugo 87 (Talk | contribs)

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.

RBS1.jpg
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.

26 07 2010 Rbs.png
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.
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.