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

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(Difference between revisions)
(Experimental setup)
(The Reference Construct)
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====The Reference Construct====
====The Reference Construct====
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[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 <i>E.coli</i> TOP10 yielded positives as analyzed by UV-light.
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====The BioBricks====
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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.
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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.
[[Image:RBS1.jpg|left|650px]]
[[Image:RBS1.jpg|left|650px]]

Revision as of 10:41, 24 October 2010

CharacterizationResultsParts

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

BBa_J23100, which is contained in the pSB1A2 plasmid was digested using SpeI and PstI. BBa_E0240 was digested using XbaI and PstI and ligated with the digest product of J23100. Transformation into E.coli TOP10 yielded positives as analyzed by UV-light.

The BioBricks

The Biobricks generated for the measurements were: K398500, K398501, K398502, K398503, K398504 and K173000. A general example 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

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