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

From 2010.igem.org

BioBricks, the making of the RBS Characterization

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: <partinfo>K398500 SpecifiedComponents</partinfo>

<partinfo>K398501 SpecifiedComponents</partinfo>

<partinfo>K398502 SpecifiedComponents</partinfo>

<partinfo>K398503 SpecifiedComponents</partinfo>

<partinfo>K398504 SpecifiedComponents</partinfo>

Experimental Setup

We took five different RBS sequences from the [http://partsregistry.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Anderson Anderson RBS family] (J61100, J61101, J61107, J61117, J61127). The standard RBS [http://partsregistry.org/Part:BBa_B0032 B0032] was used as a reference for our characterization.

All these RBS sequences were placed in front of the standard [http://partsregistry.org/Part:BBa_E0040 GFP coding sequence], so expression could be measured. The Biobricks generated in order to perform the experiments were: [http://partsregistry.org/Part:BBa_K398500 K398500], [http://partsregistry.org/Part:BBa_K398501 K398501], [http://partsregistry.org/Part:BBa_K398502 K398502], [http://partsregistry.org/Part:BBa_K398503 K398503], [http://partsregistry.org/Part:BBa_K398504 K398504]. The general map of the construction is shown below, where the RBS is displayed in fucsia.

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