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Team:TU Delft/Project/rbs-characterization/parts
From 2010.igem.org
RBS measurement parts
Experimental setup
The following RBS sequences from the Anderson RBS family were used:
For the purpose of comparison and standardization the following RBS was used as a reference for our characterization.
These RBS sequences were positioned upstream of the standard GFP coding sequence, in order to be able to measure expression.
Cloning method
The Anderson Constructs
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: K398500, K398501, K398502, K398503, K398504. A general example map of the construction is shown below, where the RBS is displayed in fucsia.
| 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 |
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>
