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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 Firefly luciferase 1.8 Reporter construct 2 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.

virA receptor

Actually we wanted to use the virA receptor already existing in the partsregistry (BBa_K238008). 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.

vir-promotor

We wanted to use the vir-promotor from the partsregistry (BBa_K238011) 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.

Neomycin / kanamycin resistance

A neomycin / kanamycin resistance gene without promotor is isolated and brought into a BioBrick compatible form.

BioBrick for virA-screenings

This part contains our mutated virG BioBrick under the control of a constitutive promotor (BBa_J23110) and an antibiotic resistance (tetracyclin or neomycin) under the control of the virB promotor. The better the virA receptor recognizes a substance the stronger will the antibiotic resistance be expressed.

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.

Firefly luciferase

Bringing the firefly luciferase gene from Promega's pGL4 vector into a BioBrick compatible form as a sensitive reporter gene.

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.