Latest revision as of 18:28, 25 October 2010

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Unfinished BioBricks

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 and working, but we will not submit it to the registry because we did not finish the R6K plasmid in a nice way.

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).

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 =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.
-You can find this BioBrick under the control of a constitutive promotor (<partinfo>J23110</partinfo>) here: <partinfo>K389010</partinfo>. This BioBrick is also finished but not submitted to the registry yet.
-===''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''-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''-promoter 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 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===
@@ Line 50: / Line 29: @@
 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.
+The screening system is finished so far and working, but we will not submit it to the registry because we did not finish the R6K plasmid in a nice way.
-===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 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 ''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.
+This BioBrick contains a ''lacI<sup>q</sup>'' and a ''lac'' operator. So the ''lac'' operator is inducible with IPTG, but tightly regulated, also in strains without ''lacI<sup>q</sup>'' (''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.

Team:Bielefeld-Germany/Results/Unfinished

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