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

From 2010.igem.org

Revision as of 11:07, 24 October 2010

RBS measurement parts

Experimental setup

The following RBS sequences from the [http://partsregistry.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Anderson Anderson RBS family] were used:

• [http://partsregistry.org/Part:BBa_J61100 BBa_J61100]
• [http://partsregistry.org/Part:BBa_J61101 BBa_J61101]
• [http://partsregistry.org/Part:BBa_J61107 BBa_J61107]
• [http://partsregistry.org/Part:BBa_J61117 BBa_J61117]
• [http://partsregistry.org/Part:BBa_J61127 BBa_J61127]

For the purpose of comparison and standardization the following RBS was used as a reference for our characterization.

• [http://partsregistry.org/Part:BBa_B0032 BBa_B0032]

These RBS sequences were positioned upstream of the standard [http://partsregistry.org/Part:BBa_E0040 GFP coding sequence], in order to be able to measure expression.

Cloning method

This is a summary of the cloning methods that succeeded. For more information on the exact methods and all attempts made see what was done under the header 'Characterization of RBS sequences' in our blogs of June and July 2010.

The Anderson Constructs

[http://partsregistry.org/Part:BBa_I13401 BBa_I13401] was PCR amplified by using the universal primers G00100 and G00101. The purified PCR product was back-digested using XbaI and PstI. The Anderson RBSs are provided in pSB1A2 plasmids. These were digested using SpeI and PstI and ligated with the digested I13401 insert. Transformation into competent TOP10 E.coli cells yielded positives, as determined by UV-light plate analysis.

The Reference Construct

[http://partsregistry.org/Part:BBa_J23100 BBa_J23100], which is contained in the pSB1A2 plasmid was digested using SpeI and PstI. [http://partsregistry.org/Part:BBa_E0240 BBa_E0240] was digested using XbaI and PstI and ligated with the digest product of J23100. Transformation into E.coli TOP10 yielded positives as determined by UV-light.

The BioBricks

The Biobricks generated for the measurements 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] and [http://partsregistry.org/Part:BBa_K173000 K173000]. A general example map of the construction is shown below, where the RBS is displayed in fucsia. Notice the additional XbaI site located between the promoter and RBS sequence. This site is inherent to the Anderson RBS inserts on the distribution plates and was maintained in our constructs in case promoter swapping would be necessary in future.