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From 2010.igem.org

Contents 1 Unfinished BioBricks 1.1 Mutated virG 1.2 virA receptor 1.3 vir-promotor 1.4 Neomycin / kanamycin resistance 1.5 BioBrick for virA-screenings 1.6 Mutated virA 1.7 Plasmid with R6K origin of replication 2 virA screening system 2.1 Firefly luciferase 2.2 Reporter construct 3 Literature

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. The virA will be under the control of a constitutive promotor (<partinfo>J23110</partinfo>).

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.

virA screening system

We will use the BioBrick for virA-screenings in a plasmid with R6K ori and the mutated virA in the pSB1C3 plasmid with ColE1 ori. Both plasmids will be installed and screened in E. coli EC100D. Once we will find a construct with high sensitivity for a screened substance, we will isolate the plasmids and transform them to e.g. E. coli TOP10. Because the R6K ori does not work in this strain, we can easily separate the mutated virA BioBrick from the screening plasmid. Because the virA is in the pSB1C3 vector, we won't have to do further cloning.

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.