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From 2010.igem.org

Contents 1 Unfinished BioBricks 1.1 Neomycin / kanamycin resistance 1.2 BioBrick for virA-screenings 1.3 Mutated virA 1.4 Plasmid with R6K origin of replication 1.5 virA screening system 1.6 Firefly luciferase 1.7 Reporter construct 1.8 Super tight controlled lac operator 2 Literature

Unfinished BioBricks

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>

This BioBrick is finished, but we have not submitted it to the registry yet

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>

This BioBrick is finished but we haven't submitted it to the registry yet.

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 promoter (<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.

This BioBrick is somehow finished - it works, but we reached our goal in a different way...

virA screening system

We will use the BioBrick for virA-screenings (<partinfo>K389011</partinfo>) in a plasmid with R6K ori and the mutated virA in the pSB1C3 plasmid with ColE1 ori. Both plasmids will be transformed to 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.

The screening system is finished so far - we are waiting for the competent cells to grow.

Firefly luciferase

Bringing the firefly luciferase gene from Promega's pGL4.10[luc2] vector into a BioBrick compatible form as a sensitive reporter gene.

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

This BioBrick is finished, but we have not submitted it to the registry yet

Reporter construct

The reporter construct is similar to the virA screening construct but instead of the antibiotic resistance it carries a reporter gene (mRFP: <partinfo>K389013</partinfo> or luciferase: <partinfo>K389012</partinfo>). 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 (<partinfo>K389411</partinfo>, <partinfo>K389412</partinfo>, <partinfo>K389413</partinfo>).

These BioBricks are finished but we haven't submitted them to the registry yet.

Super tight controlled lac operator

This BioBrick contains a lacI^q and a lac operator. So the lac operator is inducible with IPTG, but tightly regulated, also in strains without lacI^q (e.g. E. coli TOP10). This BioBrick should be used to demonstrate the sensitivity of the luciferase reporter gene compared to mRFP.

You can find this BioBrick here: <partinfo>K389050</partinfo>.

This BioBrick is finished but we haven't submitted it to the registry yet. It seems not to work as expected, though (high basal expression).

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.