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Team:Bielefeld-Germany/Results/Unfinished
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===Plasmid with R6K origin of replication=== | ===Plasmid with R6K origin of replication=== | ||
| - | In order to install our ''virA'' screening system, we need two plasmids in one cell. So we have to create a BioBrick compliant plasmid which has a different compatibility group as the standard pSBXXX plasmids. Our plan is to replace the ColE1 ori from a pSBXXX plasmid with a R6K ori. The R6K ori works ''e.g.'' in ''E. coli'' EC100D strains and is compatible with ColE1 oris. | + | In order to install our ''virA'' screening system, we need two plasmids in one cell. So we have to create a BioBrick compliant plasmid which has a different compatibility group as the standard pSBXXX plasmids. Our plan is to replace the ColE1 ori from a pSBXXX plasmid with a R6K ori (<partinfo>J61001</partinfo>). The R6K ori works ''e.g.'' in ''E. coli'' EC100D strains and is compatible with ColE1 oris. |
===Firefly luciferase=== | ===Firefly luciferase=== | ||
Revision as of 18:12, 11 August 2010
Contents |
Unfinished BioBricks
Mutated virG
This version of virG activates vir promotors in Escherichia coli without the rpoA-gene from Agrobacterium tumefaciens. For this reason the point mutations G56V and I77V are brought into the molecule (compare YC Jung et al., 2004). Because this BioBrick is synthesized by Mr. Gene, codon usage is optimized for E. coli and illegal restriction sites were removed.
You can find this BioBrick here: <partinfo>K389002</partinfo>
virA receptor
Actually we wanted to use the virA receptor already existing in the partsregistry (<partinfo>K238008</partinfo>). But due to some problems (compare results in BioBricks/tested) we decided to isolate the virA gene from A. tumefaciens C58 ourselves and bring it into a BioBrick compatible form. We removed an illegal PstI restriction site in the virA gene by site-directed mutagenesis.
You can find this BioBrick here: <partinfo>K389001</partinfo>
vir-promotor
We wanted to use the vir-promotor from the partsregistry (<partinfo>K238011</partinfo>) but the same problems occurred like with the use of the virA receptor from the partsregistry. So we also have to create a new vir-promotor BioBrick (again from A. tumefaciens C58).
You can find this BioBrick here: <partinfo>K389003</partinfo>
Neomycin / kanamycin resistance
A neomycin / kanamycin resistance gene without promotor is isolated and brought into a BioBrick compatible form. We will use the BioBrick <partinfo>P1003</partinfo> as source for the kanamycin resistance gene.
You can find this BioBrick here: <partinfo>K389004</partinfo>
BioBrick for virA-screenings
This part contains our mutated virG BioBrick under the control of a constitutive promotor (<partinfo>J23110</partinfo>) and an antibiotic resistance (<partinfo>K389004</partinfo>) under the control of the virB promotor (<partinfo>K389003</partinfo>). The better the virA receptor recognizes a substance the stronger will the antibiotic resistance be expressed.
You can find this BioBrick here: <partinfo>K389011</partinfo>
Mutated virA
The virA gene is mutated by error prone PCR and screened for several phenolic substances (e.g. capsaicin and homovanillic acid). The goal is to receive highly specific and sensitive mutated virA receptors by directed evolution.
Plasmid with R6K origin of replication
In order to install our virA screening system, we need two plasmids in one cell. So we have to create a BioBrick compliant plasmid which has a different compatibility group as the standard pSBXXX plasmids. Our plan is to replace the ColE1 ori from a pSBXXX plasmid with a R6K ori (<partinfo>J61001</partinfo>). The R6K ori works e.g. in E. coli EC100D strains and is compatible with ColE1 oris.
Firefly luciferase
Bringing the firefly luciferase gene from Promega's pGL4 vector into a BioBrick compatible form as a sensitive reporter gene.
You can find this BioBrick here: <partinfo>K389005</partinfo>
Reporter construct
The reporter construct is similar to the virA screening construct but instead of the antibiotic resistance it carries a reporter gene (e.g. GFP or luciferase). The amount of produced reporter shows the activity of the virA receptor.
Literature
YC Jung et al. (2004) Mutants of Agrobacterium tumefaciens virG Gene That Activate Transcription of vir Promoter in Escherichia coli, Current Microbiol 49:334-340.
