Team:TU Delft/project/rbs characterization

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RBS Characterization

For our RBS characterization project, 5 different RBS sequences from the Anderson RBS family (J61100, J61101, J61107, J61117, J61127) and one known RBS (B0032) where placed in front of GFP and measured over 18 hours using a Gen5 fluorenscence and absorbance plate reader. 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
26 07 2010 Rbs.png
Strength was calculated by taking the mean of the ratio between the expression of known RBS (B0032) and expression of Anderson RBS over some time. Expression being the measured fluorescence divided by measured biomass (absorbance, OD). The measurements where taken from the part of the curve where optimal growth can be assumed, so from 0:40 until 2:30 The result is a simple characterization of the Anderson RBS sequences in relation to the known RBSes. The given strengths are given in comparison with the iGEM RBS standard B0034.
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.