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From 2010.igem.org

Contents 1 Submitted BioBricks 1.1 VirA receptor 1.2 Mutated virG 1.3 virB-promoter 1.4 Firefly luciferase 1.5 Neomycin / kanamycin resistance 1.6 BioBrick for virA-screenings 1.7 Reporter construct 2 Literature

Submitted BioBricks

We have submitted the following BioBricks:

VirA receptor

The VirA receptor is used by A. tumefaciens to detect acetosyringone and other phenolic substances which are secreted by plants after injury. In presence of these substances VirA phosphorylates itself and afterwards VirG, a response regulator which activates vir promoters. These promoters control genes which are used for infecting the injured plant. 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 the TI-plasmid of 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>

You can find this BioBrick under the control of a constitutive promoter (<partinfo>J23110</partinfo>) here: <partinfo>K389010</partinfo>.

Mutated virG

Phosphorylated VirG binds to vir promoters and activates them. VirG is activated by the acetosyringone receptor VirA. This version of VirG activates vir promoters in Escherichia coli without the rpoA-gene from Agrobacterium tumefaciens. For this reason the point mutations G56V and I77V are brought into the molecule (YC Jung et al., 2004). Because this BioBrick is synthesized (Mr.Gene GmbH), codon usage is optimized for E. coli and illegal restriction sites were removed. When you use this virG gene in a VirA/G signaling system you do not need <partinfo>K238010</partinfo> anymore to get the system working in E. coli.

You can find this BioBrick here: <partinfo>K389002</partinfo>

virB-promoter

Vir-promoters from A. tumefaciens are induced by phosphorylated VirG response regulators and control genes for infecting plants in their natural host. They are part of the VirA/G signal transduction system. We wanted to use the vir-promoter from the partsregistry (<partinfo>K238011</partinfo>) but the same problems occurred like with the use of the VirA receptor BioBrick from the partsregistry. So we also have to create a new vir-promoter BioBrick (again from TI-plasmid of A. tumefaciens C58).

You can find this BioBrick here: <partinfo>K389003</partinfo>

Firefly luciferase

Bringing the firefly luciferase gene from Promega's pGL4.10[luc2] vector into a BioBrick compatible form as a sensitive reporter gene. To amplify the signal of the luciferase three different sensitivity tuners are assembled before the luciferase gene. The sensitivity tuners were created in 2007 by the iGEM team from Cambridge and amplify the read-out signal.

You can find this BioBrick here: <partinfo>K389004</partinfo>

You can find this BioBrick with sensitivity tuner 1 here: <partinfo>K389401</partinfo>

You can find this BioBrick with sensitivity tuner 2 here: <partinfo>K389402</partinfo>

You can find this BioBrick with sensitivity tuner 3 here: <partinfo>K389403</partinfo>

Neomycin / kanamycin resistance

A neomycin / kanamycin resistance gene without promoter 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>K389005</partinfo>

BioBrick for virA-screenings

This part contains our mutated virG BioBrick under the control of a constitutive promoter (<partinfo>J23110</partinfo>) and an antibiotic resistance (<partinfo>K389005</partinfo>) under the control of the virB promoter (<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>

Reporter construct

The reporter construct is similar to the virA screening construct but instead of the antibiotic resistance it carries a reporter gene. The amount of produced reporter shows the activity of the VirA receptor and the vir promoter, respectively. If the original vir promoter is too weak, we will use Cambridge's sensitivity tuners to increase the output signal of our biosensor.

You can find this BioBrick with luciferase read-out here: <partinfo>K389012</partinfo>

You can find this BioBrick with mRFP read-out here: <partinfo>K389013</partinfo>

You can find this BioBrick with amplified luciferase read-out No. 1 here: <partinfo>K389411</partinfo>

You can find this BioBrick with amplified luciferase read-out No. 2 here: <partinfo>K389412</partinfo>

You can find this BioBrick with amplified luciferase read-out No. 3 here: <partinfo>K389413</partinfo>

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.