"

From 2010.igem.org

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], so expression could be measured.

Cloning method

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

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]. A general example map of the construction is shown below, where the RBS is displayed in fucsia.

Feature	Function
[http://partsregistry.org/Part:BBa_B0032 AmpR]	Ampicillin resistance
[http://partsregistry.org/Part:BBa_B0015 B0015]	Transcriptional (double) terminator
[http://partsregistry.org/Part:BBa_B0062 B0062]	Transcriptional terminator
[http://partsregistry.org/Part:BBa_E0040 E0040]	GFP
[http://partsregistry.org/Part:BBa_G00000 G00000]	Standard prefix
[http://partsregistry.org/Part:BBa_G00001 G00001]	Standard suffix
[http://partsregistry.org/Part:BBa_G00100 G00100]	VF2 primer binding site
[http://partsregistry.org/Part:BBa_G00101 G00101]	VR primer binding site
[http://partsregistry.org/Part:BBa_J61100 J61100]	RBS Anderson family
[http://partsregistry.org/Part:BBa_J23100 J23100]	Promoter