Team:Bielefeld-Germany/Results/Used

From 2010.igem.org

(Difference between revisions)
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|style="border-style: solid; border-width: 1px"| from registry
|style="border-style: solid; border-width: 1px"| from registry
|style="border-style: solid; border-width: 1px"|  
|style="border-style: solid; border-width: 1px"|  
-
* we always assembled a strong RBS
+
* we always assembled a strong RBS (AGGAG)
|-
|-
|style="border-style: solid; border-width: 1px"| ''lacI'' gene, <partinfo>C0012</partinfo>: <partinfo>C0012 SpecifiedComponents</partinfo>
|style="border-style: solid; border-width: 1px"| ''lacI'' gene, <partinfo>C0012</partinfo>: <partinfo>C0012 SpecifiedComponents</partinfo>
|style="border-style: solid; border-width: 1px"| from registry
|style="border-style: solid; border-width: 1px"| from registry
|style="border-style: solid; border-width: 1px"|  
|style="border-style: solid; border-width: 1px"|  
-
* used this to build the tightly regulated ''lac''-operon <partinfo>K389050</partinfo>
+
* this is the lac operon repressor ''lacI''
 +
* used this to build a tightly regulated ''lac''-operon <partinfo>K389050</partinfo> with low basal transcription
|-
|-
|style="border-style: solid; border-width: 1px"| mRFP, <partinfo>E1010</partinfo>: <partinfo>E1010 SpecifiedComponents</partinfo>
|style="border-style: solid; border-width: 1px"| mRFP, <partinfo>E1010</partinfo>: <partinfo>E1010 SpecifiedComponents</partinfo>
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|style="border-style: solid; border-width: 1px"| from registry
|style="border-style: solid; border-width: 1px"| from registry
|style="border-style: solid; border-width: 1px"|  
|style="border-style: solid; border-width: 1px"|  
 +
* this is a strong constitutive promoter which is located upstream of the ''lacI'' gene in ''E. coli'' lacIq strains
* used this to build the tightly regulated ''lac''-operon <partinfo>K389050</partinfo>
* used this to build the tightly regulated ''lac''-operon <partinfo>K389050</partinfo>
|-
|-
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|style="border-style: solid; border-width: 1px"| from registry
|style="border-style: solid; border-width: 1px"| from registry
|style="border-style: solid; border-width: 1px"|  
|style="border-style: solid; border-width: 1px"|  
-
* this BioBrick was used as a visible selection marker for cloning other BioBricks into the <partinfo>pSB1C3</partinfo> plasmid
+
* this BioBrick was used as a visible selection marker for cloning other BioBricks into the <partinfo>pSB1C3</partinfo> plasmid to submit them to the registry
|-
|-
|style="border-style: solid; border-width: 1px"| constitutive promoter, <partinfo>J23110</partinfo>: <partinfo>J23110 SpecifiedComponents</partinfo>
|style="border-style: solid; border-width: 1px"| constitutive promoter, <partinfo>J23110</partinfo>: <partinfo>J23110 SpecifiedComponents</partinfo>
|style="border-style: solid; border-width: 1px"| from registry
|style="border-style: solid; border-width: 1px"| from registry
|style="border-style: solid; border-width: 1px"|  
|style="border-style: solid; border-width: 1px"|  
-
* we always used a medium strong constitutive promoter from the registry to express our BioBricks
+
* we always used a medium strong constitutive promoter from the registry to express our BioBricks, so they are neither expressed too strong so the cell suffers from metabolomic stress nor too weak so there is no visible effect of our BioBricks
* this BioBrick was used ''e.g.'' in <partinfo>K389010</partinfo> or <partinfo>K389011</partinfo>
* this BioBrick was used ''e.g.'' in <partinfo>K389010</partinfo> or <partinfo>K389011</partinfo>
|-
|-
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|style="border-style: solid; border-width: 1px"|  
|style="border-style: solid; border-width: 1px"|  
* this BioBrick was used to create a plasmid without ColE1 ori
* this BioBrick was used to create a plasmid without ColE1 ori
-
* the R6K ori only works in some ''E. coli'' strains, so it is used for a two plasmid screening system
+
* the R6K ori only works in pir+ / pir116 ''E. coli'' strains, so it is used for a two plasmid screening system (other compatibility class than ColE1 ori, easy separation of the two plasmids)
 +
* the R6K ori is a medium copy plasmid in pir+ / pir116 ''E. coli'' strains like EC100D
|-
|-
|style="border-style: solid; border-width: 1px"| ''virA'', <partinfo>K238008</partinfo>: <partinfo>K238008 SpecifiedComponents</partinfo>
|style="border-style: solid; border-width: 1px"| ''virA'', <partinfo>K238008</partinfo>: <partinfo>K238008 SpecifiedComponents</partinfo>
|style="border-style: solid; border-width: 1px"| from registry, fixed
|style="border-style: solid; border-width: 1px"| from registry, fixed
|style="border-style: solid; border-width: 1px"|  
|style="border-style: solid; border-width: 1px"|  
-
* we wanted to use the ''virA'' BioBrick from the registry but problems occurred, so we created our own ''virA'' from ''Agrobacterium tumefaciens'' C58 TI-plasmid
+
* we wanted to use this ''virA'' BioBrick from the registry but problems occurred, so we created our own ''virA'' from ''Agrobacterium tumefaciens'' C58 TI-plasmid
* our own (working) ''virA'' is here: <partinfo>K389001</partinfo>
* our own (working) ''virA'' is here: <partinfo>K389001</partinfo>
|-
|-
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|style="border-style: solid; border-width: 1px"| from registry, fixed
|style="border-style: solid; border-width: 1px"| from registry, fixed
|style="border-style: solid; border-width: 1px"|  
|style="border-style: solid; border-width: 1px"|  
-
* same as the ''virA'' BioBrick - this one from the registry does not work properly, so we made a ''vir''-promoter by ourselves
+
* same as the ''virA'' BioBrick - this one from the registry does not work properly, so we made a ''vir''-promoter by ourselves (<partinfo>K389003</partinfo>)
* the part that was sent to us is actually a ''tetR'' gene (<partinfo>C0040</partinfo>) under the control of a constitutive promoter (<partinfo>J23105</partinfo>) before a double terminator (<partinfo>B0015</partinfo>, compare our sequencing results)
* the part that was sent to us is actually a ''tetR'' gene (<partinfo>C0040</partinfo>) under the control of a constitutive promoter (<partinfo>J23105</partinfo>) before a double terminator (<partinfo>B0015</partinfo>, compare our sequencing results)
|-
|-
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* illegal PstI restriction site removed by site-directed mutagenesis
* illegal PstI restriction site removed by site-directed mutagenesis
* isolated from ''A. tumefaciens'' C58 TI-plasmid
* isolated from ''A. tumefaciens'' C58 TI-plasmid
 +
* functionality of this BioBrick is prooved with <partinfo>K389015</partinfo> and <partinfo>K389016</partinfo>
|-
|-
|style="border-style: solid; border-width: 1px"| ''virG'', <partinfo>K389002</partinfo>: <partinfo>K389002 SpecifiedComponents</partinfo>
|style="border-style: solid; border-width: 1px"| ''virG'', <partinfo>K389002</partinfo>: <partinfo>K389002 SpecifiedComponents</partinfo>

Revision as of 12:31, 12 October 2010

{{{1}}}

Used BioBricks

This is a list of all BioBricks we used from the registry or created by ourselves and a short explanation, why we used them. The list is sorted alphabetically.

BioBrick (name and partnumber) Status Comment
double terminator, <partinfo>B0017</partinfo>: <partinfo>B0017 SpecifiedComponents</partinfo> from registry
  • this contains two <partinfo>B0010</partinfo> terminators
  • we used this because of problems mentioned in registry with <partinfo>B0012</partinfo>
RBS, <partinfo>B0034</partinfo>: <partinfo>B0034 SpecifiedComponents</partinfo> from registry
  • we always assembled a strong RBS (AGGAG)
lacI gene, <partinfo>C0012</partinfo>: <partinfo>C0012 SpecifiedComponents</partinfo> from registry
  • this is the lac operon repressor lacI
  • used this to build a tightly regulated lac-operon <partinfo>K389050</partinfo> with low basal transcription
mRFP, <partinfo>E1010</partinfo>: <partinfo>E1010 SpecifiedComponents</partinfo> from registry
  • this BioBrick was used as a reporter gene, e.g. in the construct <partinfo>K389013</partinfo>
lacIq promoter, <partinfo>I14032</partinfo>: <partinfo>I14032 SpecifiedComponents</partinfo> from registry
  • this is a strong constitutive promoter which is located upstream of the lacI gene in E. coli lacIq strains
  • used this to build the tightly regulated lac-operon <partinfo>K389050</partinfo>
sensitivity tuner, <partinfo>I746370</partinfo>: <partinfo>I746370 SpecifiedComponents</partinfo> from registry
  • sensitivity tuner 1...
sensitivity tuner, <partinfo>I746380</partinfo>: <partinfo>I746380 SpecifiedComponents</partinfo> from registry
  • sensitivity tuner 2...
sensitivity tuner, <partinfo>I746390</partinfo>: <partinfo>I746390 SpecifiedComponents</partinfo> from registry
  • sensitivity tuner 3...
mRFP generator, <partinfo>J04450</partinfo>: <partinfo>J04450 SpecifiedComponents</partinfo> from registry
  • this BioBrick was used as a visible selection marker for cloning other BioBricks into the <partinfo>pSB1C3</partinfo> plasmid to submit them to the registry
constitutive promoter, <partinfo>J23110</partinfo>: <partinfo>J23110 SpecifiedComponents</partinfo> from registry
  • we always used a medium strong constitutive promoter from the registry to express our BioBricks, so they are neither expressed too strong so the cell suffers from metabolomic stress nor too weak so there is no visible effect of our BioBricks
  • this BioBrick was used e.g. in <partinfo>K389010</partinfo> or <partinfo>K389011</partinfo>
R6K origin, <partinfo>J61001</partinfo>: <partinfo>J61001 SpecifiedComponents</partinfo> from registry
  • this BioBrick was used to create a plasmid without ColE1 ori
  • the R6K ori only works in pir+ / pir116 E. coli strains, so it is used for a two plasmid screening system (other compatibility class than ColE1 ori, easy separation of the two plasmids)
  • the R6K ori is a medium copy plasmid in pir+ / pir116 E. coli strains like EC100D
virA, <partinfo>K238008</partinfo>: <partinfo>K238008 SpecifiedComponents</partinfo> from registry, fixed
  • we wanted to use this virA BioBrick from the registry but problems occurred, so we created our own virA from Agrobacterium tumefaciens C58 TI-plasmid
  • our own (working) virA is here: <partinfo>K389001</partinfo>
vir-promoter, <partinfo>K238011</partinfo>: <partinfo>K238011 SpecifiedComponents</partinfo> from registry, fixed
  • same as the virA BioBrick - this one from the registry does not work properly, so we made a vir-promoter by ourselves (<partinfo>K389003</partinfo>)
  • the part that was sent to us is actually a tetR gene (<partinfo>C0040</partinfo>) under the control of a constitutive promoter (<partinfo>J23105</partinfo>) before a double terminator (<partinfo>B0015</partinfo>, compare our sequencing results)
virA, <partinfo>K389001</partinfo>: <partinfo>K389001 SpecifiedComponents</partinfo> new
  • "our" virA
  • illegal PstI restriction site removed by site-directed mutagenesis
  • isolated from A. tumefaciens C58 TI-plasmid
  • functionality of this BioBrick is prooved with <partinfo>K389015</partinfo> and <partinfo>K389016</partinfo>
virG, <partinfo>K389002</partinfo>: <partinfo>K389002 SpecifiedComponents</partinfo> synthesized, new
  • virG that works in E. coli without rpoA gene from A. tumefaciens
  • mutations ... + optimized codon-usage (E. coli) + removal of all illegal restriction sites because it was synthesized
vir-promoter, <partinfo>K389003</partinfo>: <partinfo>K389003 SpecifiedComponents</partinfo> new
  • "our" vir-promoter
  • is induced by phosphorylated VirG
  • isolated from A. tumefaciens C58 TI-plasmid
firefly luciferase, <partinfo>K389004</partinfo>: <partinfo>K389004 SpecifiedComponents</partinfo> new
  • "our" luciferase
  • sensitive reporter gene
  • isolated from Promega's pGL4.10luc2 plasmid
Kanamycin resistance gene, <partinfo>K389005</partinfo>: <partinfo>K389005 SpecifiedComponents</partinfo> new
  • "our" kanamycin / neomycin resistance
  • used for directed evolution of virA and mutated virA screenings, respectively
  • isolated from the BioBrick <partinfo>P1003</partinfo>
virA generator, <partinfo>K389010</partinfo>: <partinfo>K389010 SpecifiedComponents</partinfo> composite
  • medium strong constitutive promoter, so there is enough VirA receptor expressed but not too much so the cell suffers from the expression
  • this version exists in a <partinfo>pSB1C3</partinfo> and a <partinfo>pSB1AT3</partinfo> vector
virA screening device, <partinfo>K389011</partinfo>: <partinfo>K389011 SpecifiedComponents</partinfo> composite
  • the new virG which works without rpoA is expressed constitutively and a kanamycin resistance gene is under the control of a vir promoter to screen mutants of virA (which are on a different plasmid in a <partinfo>K389010</partinfo>-like part)
  • the better the VirA receptor recognizes a substance the more resistant the cell is against kanamyin
  • this device has to be on a plasmid with different ori than the virA which should be screened (different compatibility classes)
  • we cloned this BioBrick into a plasmid with R6K ori which only works in pir+ or pir116 strains (e.g. E. coli EC100D), so we can easily seperate the two plasmids by transforming them into strains without Pir protein (e.g. E. coli TOP10)
virA reporter device with luc, <partinfo>K389012</partinfo>: <partinfo>K389012 SpecifiedComponents</partinfo> composite
  • this BioBrick is similar to <partinfo>K389011</partinfo> but with the reporter luciferase instead of an antibiotic resistance gene
  • with this BioBrick it is possible to measure the activity of the vir promoter and the VirA receptor, respectively
virA reporter device with mRFP, <partinfo>K389013</partinfo>: <partinfo>K389013 SpecifiedComponents</partinfo> composite
  • this BioBrick is similar to <partinfo>K389011</partinfo> but with the reporter mRFP instead of an antibiotic resistance gene
  • with this BioBrick it is possible to measure the activity of the vir promoter and the VirA receptor, respectively
Test, <partinfo>K389014</partinfo>: <partinfo>K389014 SpecifiedComponents</partinfo> composite
  • this BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo>
  • used for testing the general screening concept of expressing an antibiotic resistance with a vir promoter after induction with acetosyringone (the natural VirA inductor)
vir promoter characterisation part with luc, <partinfo>K389015</partinfo>: <partinfo>K389015 SpecifiedComponents</partinfo> composite
  • this BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389012</partinfo>
  • used for characterizing the natural VirA/G system and the vir promoter, respectively
vir promoter characterisation part with mRFP, <partinfo>K389016</partinfo>: <partinfo>K389016 SpecifiedComponents</partinfo> composite
  • this BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389013</partinfo>
  • used for characterizing the natural VirA/G system and the vir promoter, respectively
vir promoter part, <partinfo>K389017</partinfo>: <partinfo>K389017 SpecifiedComponents</partinfo> composite
  • this is a complete VirA/G signaling system without a reporter gene, so any gene of interest can be assembled to a vir promoter
tightly controlled lac operator, <partinfo>K389050</partinfo>: <partinfo>K389050 SpecifiedComponents</partinfo> composite
  • a lac operator is combined with a lacIq so there is always enough lacI to repress the lac operator
  • the system is still inducible with IPTG but it has a low basal transcription
  • this part is used to test the sensitivity of the luciferase BioBrick <partinfo>K389004</partinfo> and compare it to the sensitivity of the mRFP BioBrick <partinfo>E1010</partinfo>
tightly controlled lac operator + luc, <partinfo>K389051</partinfo>: <partinfo>K389051 SpecifiedComponents</partinfo> composite
  • a lac operator is combined with a lacIq so there is always enough lacI to repress the lac operator
  • the system is still inducible with IPTG but it has a low basal transcription
  • ...
tightly controlled lac operator + mRFP, <partinfo>K389052</partinfo>: <partinfo>K389052 SpecifiedComponents</partinfo> composite
  • a lac operator is combined with a lacIq so there is always enough lacI to repress the lac operator
  • the system is still inducible with IPTG but it has a low basal transcription
  • ...
sensitivity tuner no. 1 without promoter + luc readout, <partinfo>K389401</partinfo>: <partinfo>K389401 SpecifiedComponents</partinfo> new / modified
  • this is a modified <partinfo>I746370</partinfo> sensitivity tuner
  • we erased the arabinose promoter and the GFP readout from the original part and added our firefly luciferase BioBrick as a new readout
  • this part can be used behind a random promoter and gives an enhanced luciferase readout signal
sensitivity tuner no. 2 without promoter + luc readout, <partinfo>K389402</partinfo>: <partinfo>K389402 SpecifiedComponents</partinfo> new / modified
  • this is a modified <partinfo>I746380</partinfo> sensitivity tuner
  • we erased the arabinose promoter and the GFP readout from the original part and added our firefly luciferase BioBrick as a new readout
  • this part can be used behind a random promoter and gives an enhanced luciferase readout signal
sensitivity tuner no. 3 without promoter + luc readout, <partinfo>K389403</partinfo>: <partinfo>K389403 SpecifiedComponents</partinfo> new / modified
  • this is a modified <partinfo>I746390</partinfo> sensitivity tuner
  • we erased the arabinose promoter and the GFP readout from the original part and added our firefly luciferase BioBrick as a new readout
  • this part can be used behind a random promoter and gives an enhanced luciferase readout signal
virA reporter device with sensitivity tuner no. 1 + luc readout, <partinfo>K389411</partinfo>: <partinfo>K389411 SpecifiedComponents</partinfo> composite
  • this is a modified <partinfo>I746370</partinfo> sensitivity tuner + luciferase readout behind a vir promoter
  • this part also contains a virG gene under the control of a constitutive promoter
  • in front of this part a mutated virA from a <partinfo>K389010</partinfo>-like BioBrick is assembled to get a strong readout signal in order to our goal to make spiciness visible
virA reporter device with sensitivity tuner no. 2 + luc readout, <partinfo>K389412</partinfo>: <partinfo>K389412 SpecifiedComponents</partinfo> composite
  • this is a modified <partinfo>I746380</partinfo> sensitivity tuner + luciferase readout behind a vir promoter
  • this part also contains a virG gene under the control of a constitutive promoter
  • in front of this part a mutated virA from a <partinfo>K389010</partinfo>-like BioBrick is assembled to get a strong readout signal in order to our goal to make spiciness visible
virA reporter device with sensitivity tuner no. 3 + luc readout, <partinfo>K389413</partinfo>: <partinfo>K389413 SpecifiedComponents</partinfo> composite
  • this is a modified <partinfo>I746390</partinfo> sensitivity tuner + luciferase readout behind a vir promoter
  • this part also contains a virG gene under the control of a constitutive promoter
  • in front of this part a mutated virA from a <partinfo>K389010</partinfo>-like BioBrick is assembled to get a strong readout signal in order to our goal to make spiciness visible
vir promoter characterisation part with sensitivity tuner no. 1 + luc readout, <partinfo>K389421</partinfo>: <partinfo>K389421 SpecifiedComponents</partinfo> composite
  • this is a modified <partinfo>I746370</partinfo> sensitivity tuner + luciferase readout behind a vir promoter characterisation BioBrick
  • this part also contains a virG and an unmutated virA gene under the control of a constitutive promoter
  • this BioBrick is used to proove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible
vir promoter characterisation part with sensitivity tuner no. 2 + luc readout, <partinfo>K389422</partinfo>: <partinfo>K389422 SpecifiedComponents</partinfo> composite
  • this is a modified <partinfo>I746380</partinfo> sensitivity tuner + luciferase readout behind a vir promoter characterisation BioBrick
  • this part also contains a virG and an unmutated virA gene under the control of a constitutive promoter
  • this BioBrick is used to proove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible
vir promoter characterisation part with sensitivity tuner no. 3 + luc readout, <partinfo>K389423</partinfo>: <partinfo>K389423 SpecifiedComponents</partinfo> composite
  • this is a modified <partinfo>I746390</partinfo> sensitivity tuner + luciferase readout behind a vir promoter characterisation BioBrick
  • this part also contains a virG and an unmutated virA gene under the control of a constitutive promoter
  • this BioBrick is used to proove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible
Kanamycin-resistance cassette, <partinfo>P1003</partinfo>: <partinfo>P1003 SpecifiedComponents</partinfo> from registry
  • we used this BioBrick to create <partinfo>K389005</partinfo>
ccdB-gene, <partinfo>P1010</partinfo>: <partinfo>P1010 SpecifiedComponents</partinfo> from registry
  • we used this BioBrick for 3A-assemblies
high-copy plasmid with Amp and Tet resistance, <partinfo>pSB1AT3</partinfo>: <partinfo>pSB1AT3 SpecifiedComponents</partinfo> from registry
  • we used this plasmids in 3A-assemblies and as the backbone of the mutated and unmutated <partinfo>K389010</partinfo> part
high-copy plasmid with Cm resistance, <partinfo>pSB1C3</partinfo>: <partinfo>pSB1C3 SpecifiedComponents</partinfo> from registry
  • the plasmid to send in the BioBricks
lac operator, <partinfo>R0010</partinfo>: <partinfo>R0010 SpecifiedComponents</partinfo> from registry
  • used to build a tightly regulated lac operator (<partinfo>K389050</partinfo>)
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