Team:Bielefeld-Germany/Results/Used

From 2010.igem.org

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* "our" antibiotic resistance
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* "our" kanamycin / neomycin resistance
* used for directed evolution of ''virA'' and mutated ''virA'' screenings, respectively
* used for directed evolution of ''virA'' and mutated ''virA'' screenings, respectively
* isolated from the BioBrick <partinfo>P1003</partinfo>
* isolated from the BioBrick <partinfo>P1003</partinfo>
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* medium strong constitutive promoter, so there is enough VirA receptor expressed but not too much so the cell suffers from the expression
* medium strong constitutive promoter, so there is enough VirA receptor expressed but not too much so the cell suffers from the expression
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* ...
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* this version exists in a <partinfo>pSB1C3</partinfo> and a <partinfo>pSB1AT3</partinfo> vector
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|style="border-style: solid; border-width: 1px"| ''virA'' screening device, <partinfo>K389011</partinfo>
|style="border-style: solid; border-width: 1px"| ''virA'' screening device, <partinfo>K389011</partinfo>
|style="border-style: solid; border-width: 1px"| composite
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* the new ''virG'' which works without ''rpoA'' is expressed constitutively and a kanamycin resistance is gene 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 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
* 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)
* this device has to be on a plasmid with different ori than the ''virA'' which should be screened (different compatibility classes)
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* ...
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* this BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389012</partinfo>
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* used for characterizing the natural VirA/G system and the ''vir'' promoter, respectively
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|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with mRFP, <partinfo>K389016</partinfo>
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with mRFP, <partinfo>K389016</partinfo>
|style="border-style: solid; border-width: 1px"| composite
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* ...
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* this BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389013</partinfo>
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* used for characterizing the natural VirA/G system and the ''vir'' promoter, respectively
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|style="border-style: solid; border-width: 1px"| ''vir'' promoter part, <partinfo>K389017</partinfo>
|style="border-style: solid; border-width: 1px"| ''vir'' promoter part, <partinfo>K389017</partinfo>
|style="border-style: solid; border-width: 1px"| composite
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* ...
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* this is a complete VirA/G signaling system without a reporter gene, so any gene of interest can be assembled to a ''vir'' promoter
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|style="border-style: solid; border-width: 1px"| tightly controlled ''lac'' operator, <partinfo>K389050</partinfo>
|style="border-style: solid; border-width: 1px"| tightly controlled ''lac'' operator, <partinfo>K389050</partinfo>
|style="border-style: solid; border-width: 1px"| composite
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* ...
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* a lac operator is combined with a lacIq so there is always enough lacI to repress the lac operator
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* the system is still inducible with IPTG but it has a low basal transcription
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* 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>
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|style="border-style: solid; border-width: 1px"| Kanamycin-resistance cassette, <partinfo>P1003</partinfo>
|style="border-style: solid; border-width: 1px"| Kanamycin-resistance cassette, <partinfo>P1003</partinfo>

Revision as of 12:09, 7 October 2010

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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> 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> from registry
  • we always assembled a strong RBS
lacI gene, <partinfo>C0012</partinfo> from registry
  • used this to build the tightly regulated lac-operon <partinfo>K389050</partinfo>
mRFP, <partinfo>E1010</partinfo> from registry
  • this BioBrick was used as a reporter gene, e.g. in the construct <partinfo>K389013</partinfo>
lacIq promoter, <partinfo>I14032</partinfo> from registry
  • used this to build the tightly regulated lac-operon <partinfo>K389050</partinfo>
mRFP generator, <partinfo>J04450</partinfo> from registry
  • this BioBrick was used as a visible selection marker for cloning other BioBricks into the <partinfo>pSB1C3</partinfo> plasmid
constitutive promoter, <partinfo>J23110</partinfo> from registry
  • we always used a medium strong constitutive promoter from the registry to express our BioBricks
  • this BioBrick was used e.g. in <partinfo>K389010</partinfo> or <partinfo>K389011</partinfo>
R6K origin, <partinfo>J61001</partinfo> from registry
  • 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
virA, <partinfo>K238008</partinfo> from registry, fixed
  • 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
  • our own (working) virA is here: <partinfo>K389001</partinfo>
vir-promoter, <partinfo>K238011</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
  • 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> new
  • "our" virA
  • illegal PstI restriction site removed by site-directed mutagenesis
  • isolated from A. tumefaciens C58 TI-plasmid
virG, <partinfo>K389002</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> new
  • "our" vir-promoter
  • is induced by phosphorylated VirG
  • isolated from A. tumefaciens C58 TI-plasmid
firefly luciferase, <partinfo>K389004</partinfo> new
  • "our" luciferase
  • sensitive reporter gene
  • isolated from Promega's pGL4.10luc2 plasmid
Kanamycin resistance gene, <partinfo>K389005</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> 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> 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> 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> 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> 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> 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> 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> 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> 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>
Kanamycin-resistance cassette, <partinfo>P1003</partinfo> from registry
  • we used this BioBrick to create <partinfo>K389005</partinfo>
ccdB-gene, <partinfo>P1010</partinfo> from registry
  • we used this BioBrick for 3A-assemblies
high-copy plasmid with Amp and Tet resistance, <partinfo>pSB1AT3</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> from registry
  • the plasmid to send in the BioBricks
lac operator, <partinfo>R0010</partinfo> from registry
  • used to build a tightly regulated lac operator (<partinfo>K389050</partinfo>)
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