"
Team:Bielefeld-Germany/Results/Unfinished
From 2010.igem.org
(→Reporter construct) |
|||
| Line 56: | Line 56: | ||
===Reporter construct=== | ===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. If the original ''vir'' promotor is too weak, we will use Cambridge's sensitivity tuners to increase the output signal of our biosensor. | 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. If the original ''vir'' promotor is too weak, we will use Cambridge's sensitivity tuners to increase the output signal of our biosensor. | ||
| + | |||
| + | ===Super tight controlled ''lac'' operator=== | ||
| + | This BioBrick contains a ''lacIq'' and a ''lac'' operator. So the ''lac'' operator is inducible with IPTG, but tightly regulated, also in strains without ''lacIq'' (''e.g.'' ''E. coli'' TOP10). This BioBrick should be used to demonstrate the sensitivity of the luciferase reporter gene compared to mRFP. | ||
==Literature== | ==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. | 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. | ||
Revision as of 13:47, 13 September 2010
Contents |
Unfinished BioBricks
Mutated virG
Phosphorylated VirG binds to vir promotors and activates them. VirG is activated by the acetosyringone receptor VirA. 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 (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>
This BioBrick is finished but we haven't submitted it to the registry yet.
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 VirG, a response regulator which activates vir promotors. These promotors 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>
This BioBrick is finished but we haven't submitted it to the registry yet.
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 TI-plasmid of A. tumefaciens C58).
You can find this BioBrick here: <partinfo>K389003</partinfo>
This BioBrick is finished but we haven't submitted it to the registry yet.
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>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 promotor (<partinfo>J23110</partinfo>) and an antibiotic resistance (<partinfo>K389005</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 (<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.
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>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. If the original vir promotor is too weak, we will use Cambridge's sensitivity tuners to increase the output signal of our biosensor.
Super tight controlled lac operator
This BioBrick contains a lacIq and a lac operator. So the lac operator is inducible with IPTG, but tightly regulated, also in strains without lacIq (e.g. E. coli TOP10). This BioBrick should be used to demonstrate the sensitivity of the luciferase reporter gene compared to mRFP.
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.
