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

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{{Team:TU_Delft/frame_check}}
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==BioBricks, the making of the RBS Characterization==
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__NOTOC__
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{{Team:TU_Delft/frame_check}}
<html><center><img src="http://2010.igem.org/wiki/images/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html>
<html><center><img src="http://2010.igem.org/wiki/images/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html>
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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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==RBS measurement parts==
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Transformation into competent Top10 E.coli cells yielded positives, as determined by fluorescence analysis.
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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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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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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], in order to be able to measure expression.
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{{Team:TU_Delft/frame_check}}
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===Cloning method===
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== Parts ==
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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 blog posts starting around
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Our constructs used for measurements:
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[http://2010.igem.org/Team:TU_Delft#page=Notebook/blog&blog=6_July_2010 6 July]
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<partinfo>K398500 SpecifiedComponents</partinfo>
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<partinfo>K398501 SpecifiedComponents</partinfo>
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====The Anderson Constructs====
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[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.
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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 <i>E.coli</i> cells yielded positives, as determined by UV-light plate analysis.
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<partinfo>K398502 SpecifiedComponents</partinfo>
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====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 determined by UV-light.
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<partinfo>K398503 SpecifiedComponents</partinfo>
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====The BioBricks====
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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. 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.
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<partinfo>K398504 SpecifiedComponents</partinfo>
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==Experimental Setup==
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We took five different RBS sequences from the [http://partsregistry.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Anderson Anderson RBS family] (J61100, J61101, J61107, J61117, J61127). The standard RBS [http://partsregistry.org/Part:BBa_B0032 B0032] was used as a reference for our characterization.
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All these RBS sequences were placed in front of the standard [http://partsregistry.org/Part:BBa_E0040 GFP coding sequence], so expression could be measured.
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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]]
[[Image:RBS1.jpg|left|650px]]
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===Continue Reading about the RBS Characterization===
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== Parts ==
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The constructs used for measurements:
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<partinfo>K398500 SpecifiedComponents</partinfo>
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<partinfo>K398501 SpecifiedComponents</partinfo>
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<partinfo>K398502 SpecifiedComponents</partinfo>
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<partinfo>K398503 SpecifiedComponents</partinfo>
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<partinfo>K398504 SpecifiedComponents</partinfo>
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<partinfo>K173000 SpecifiedComponents</partinfo>
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<partinfo>I13401 SpecifiedComponents</partinfo>
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<html><center><img src="http://2010.igem.org/wiki/images/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html>
<html><center><img src="http://2010.igem.org/wiki/images/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html>

Latest revision as of 20:09, 27 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

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 blog posts starting around 6 July

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 determined 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. 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.

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

The 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

J23100
GFP report
E0240
GFP
E0040

B0015

CharacterizationResultsParts