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

From 2010.igem.org

(Difference between revisions)
(Experimental Setup)
(BioBricks, the making of the RBS Characterization)
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==BioBricks, the making of the RBS Characterization==
==BioBricks, the making of the RBS Characterization==
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===Experimental setup===
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The following RBS sequences from the [http://partsregistry.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Anderson Anderson RBS family] were used:
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*[http://partsregistry.org/Part:BBa_J61100 BBa_J61100]
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*[http://partsregistry.org/Part:BBa_J61101 BBa_J61101]
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*[http://partsregistry.org/Part:BBa_J61107 BBa_J61107]
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*[http://partsregistry.org/Part:BBa_J61117 BBa_J61117]
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*[http://partsregistry.org/Part:BBa_J61127 BBa_J61127]
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For the purpose of comparison and standardization the following RBS was used as a reference for our characterization.
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*[http://partsregistry.org/Part:BBa_B0032 BBa_B0032]
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These RBS sequences were positioned upstream of the standard [http://partsregistry.org/Part:BBa_E0040 GFP coding sequence], so expression could be measured.
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.
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.
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Transformation into competent Top10 E.coli cells yielded positives, as determined by fluorescence analysis.
Transformation into competent Top10 E.coli cells yielded positives, as determined by fluorescence analysis.
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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.
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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]. The general map of the construction is shown below, where the RBS is displayed in fucsia.
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[[Image:RBS1.jpg|left|650px]]
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''Feature'''
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|'''Function'''
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|-
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|[http://partsregistry.org/Part:BBa_B0032 AmpR]
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|Ampicillin resistance
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|-
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|[http://partsregistry.org/Part:BBa_B0015 B0015]
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|Transcriptional (double) terminator
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|-
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|[http://partsregistry.org/Part:BBa_B0062 B0062]
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|Transcriptional terminator
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|-
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|[http://partsregistry.org/Part:BBa_E0040 E0040]
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|GFP
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|-
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|[http://partsregistry.org/Part:BBa_G00000 G00000]
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|Standard prefix
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|-
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|[http://partsregistry.org/Part:BBa_G00001 G00001]
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|Standard suffix
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|-
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|[http://partsregistry.org/Part:BBa_G00100 G00100]
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|VF2 primer binding site
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|-
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|[http://partsregistry.org/Part:BBa_G00101 G00101]
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|VR primer binding site
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|-
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|[http://partsregistry.org/Part:BBa_J61100 J61100]
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|RBS Anderson family
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|-
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|[http://partsregistry.org/Part:BBa_J23100 J23100]
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|Promoter
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|}
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<html><div style='clear:both'>
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</html>
== Parts ==
== Parts ==

Revision as of 14:53, 23 October 2010

CharacterizationResultsParts

BioBricks, the making of the RBS Characterization

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, so expression could be measured.

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 Biobricks generated in order to perform the experiments were: K398500, K398501, K398502, K398503, K398504. The general 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