Team:TU Delft/Project/rbs-characterization/parts

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BioBricks, the making of

BBa_I13401 was PCR amplified using the universal primers G00100 and G00101. The purified PCR product was assembled into one of the Anderson RBS plasmids provided in the distribution plates by means of 2-way ligations.

Transformation into competent Top10 E.coli cells yielded positives, as determined by fluorescence analysis.

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. Only the results obtained during exponential growth phase were taken into account.

Parts

Our constructs used for measurements:


J23100

J61100
GFP
E0040

B0015

J23100

J61101
GFP
E0040

B0015

J23100

J61107
GFP
E0040

B0015

J23100

J61117
GFP
E0040

B0015

J23100

J61127
GFP
E0040

B0015


Experimental Setup

We took five different RBS sequences from the Anderson RBS family (J61100, J61101, J61107, J61117, J61127). The standard RBS B0032 was used as a reference for our characterization.

All these RBS sequences were placed in front of the standard GFP coding sequence, so expression could be measured. 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