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Latest revision as of 03:10, 28 October 2010

The Approach

In order to create a novel biosensor we started to search for a sensor system which is able to detect certain chemicals and we looked for additional substances we could try to detect. We were looking for a system that should be taken out of a different bacteria species than Escherichia coli in order to avoid any background, when working with E. coli in the end. Therefore we checked the literature for a well reviewed sensor system, which is not part of the E. coli genome. Finally we decided to use the phenolic sensing VirA/G signaling system of Agrobacterium tumefaciens. It naturally detects acetosyringone, which is a secondary metabolite of plants that affects bacteria as an attractant. After repeated research to check for any other possible substances which could be detected by the system, we got a really long list of possibilities and picked 'capsaicin', which is responsible for the spiciness of edibles.

The biological steps for modulating the native VirA/G sensor system, in order to detect new substances are:

Extract the complete VirA/G signaling system out of A. tumefaciens
Create new BioBricks out of the existing environmental parts
Get the new BioBricks working into E. coli
Choose a reporter gene as readout for the sensor system
Modify the system for sensibility and specificity by error-prone PCR, find and select the most promising mutants (directed mutagenesis)

Preparing the system

We tried to work with the already existing VirA receptor BioBrick from the registry (<partinfo>K238008</partinfo>). Unfortunately this VirA BioBrick did not work in our experiments. We decided to sequence this BioBrick from the registry on our own and found that the actual sequence data of this BioBrick did not fit to the published sequence. Thus, in order to create this BioBrick we had to extract the virA gene via PCR out of the TI-plasmid from A. tumefaciens.

To construct the complete sensor system VirA is not enough, since it works only in combination with the VirG protein. ThevirG gene was also present in the iGEM registry, but this version containes illegal restriction sites. Moreover it needs the help of the RpoA protein from A. tumefaciens to work as a transcription facor in E. coli. In order to eliminate these drawbacks we changed the sequence manually and had the gene synthesized by Mr. Gene.

Starting point for BioBricks -> Go cloning

After discussing the possibility of creating only one big plasmid for mutated virA screenings, we unified that its size would minimize transformation efficiency and would be difficult to modify via error-prone PCR. Thereby we decided to use a system with two plasmids per cell that offers certain advantages, which will be explained later. Prior to the construction of the plasmids we had to change the origin of a pSB1C3 plasmid in order to avoid incompatibility between both plasmids. Thus we cloned the R6K origin (<partinfo>J61001</partinfo>) into the <partinfo>pSB1C3</partinfo> plasmid and subsequently removed the original pMB1/ColE1 origin of replication. Therefore we digested the pSB1C3::R6K plasmid with Hin6I and seperated the resulting 6 fragments (1758 bp, 270 bp, 174 bp, 109 bp, 100 bp, 67 bp) by agarose gel electrophoresis. We extracted the largest fragment (1758 bp) and religated it. The resulting plasmid still contains 32 remaining basepairs of the pMB1 origin, but this fragment does not enable replication in pir^- strains.

The error prone PCR and screening

After building a plasmid with R6K ori we were able to create the two constructs. The first plasmid will be found inside the competent bacteria cells and contains the virG gene under the constitutive promoter <partinfo>J23110</partinfo>, a terminator (<partinfo>B0017</partinfo>), a vir promoter and a readout or selection gene (luciferase, mRFP and kanamycin resistance, respectively):

The second plasmid contains variants of virA ,which have been altered randomly by error-prone PCR, under the control of the constitutive promoter <partinfo>J23110</partinfo>.

The error-prone PCR is a PCR using in inaccurate Taq polymerase under intesified malfunction conditions. It is possible to regulate the frequency of mutagenic events by using unbalanced concentrations of nucleotides and special salt concentrations in the PCR mixture. Using this mutagenesis we are going to create a new variants of the virA BioBrick and transform it in our cells with the read out system already present.

Survival of the fittest

We used high amounts of the antibiotic kanamycin in order to select the most specific systems for the detection of the chosen substances. By varying the amount of kanamycin we will be able to carefully select some promising clones out of thousands bacteria. The mutated virA system will be induced after the error-prone PCR by a mix of possible targets for the system (like capcaicin, dopamin, homovanillic acid etc.).

Side directed mutagenesis

Alternatively to the random mutagenesis we developed two in-silico based strategies for side directed mutagenesis as described by Looger et al..

VirA_mut1

to develop a strategy for change in the protein sequence, we used the analysis tool Muscle, which provided an advice to change one amino acid in the receptor. This conversion of leucin 293 to tyrosine was performed using site directed mutagenesis

VirA_mut2

Since VirA_mut1 with its single mutation failed working in our later screening system, we decided to mutate more amino acids in the area of the predicted binding region from animal TRPV1 receptors.

According to the literature the amino acid residues 288 to 293 of this TRPV1 receptor regulate phenol structural specificity. Substitutions in this region narrow the active phenols to those missing the para-acyl substituent (Lin et al., 2008). Our VirA_mut2 construct contains the following substitutions:

A357H *R358P *R359T *L360L *D361K *Y361L

Alignment of crucial ligand binding regions from Agrobacterium tumefaciens (lane 1), chick (lane 2), homo sapiens (lane 3), rattus (lane 4) and mus muskulus (lane 5). The Box shows all six amino acids we mutagenized in our VirA_mut2 approach

The final construct

In order to get a visible light signal by the reporter gene, we integrated the quantifiable proteins luciferase and mRFP. We also experimented with certain sensitivity tuners to be able to enhance the detected signal.

For a demonstration of this final system see the animation below:

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+= The Approach =
-== The Approach ==
+In order to create a novel biosensor we started to search for a sensor system which is able to detect certain chemicals and we looked for additional substances we could try to detect. We were looking for a system that should be taken out of a different bacteria species than ''Escherichia coli'' in order to avoid any background, when working with ''E. coli'' in the end. Therefore we checked the literature for a well reviewed sensor system, which is not part of the ''E. coli'' genome. Finally we decided to use the phenolic sensing VirA/G signaling system of ''Agrobacterium tumefaciens''. It naturally detects acetosyringone, which is a secondary metabolite of plants that affects bacteria as an attractant. After repeated research to check for any other possible substances which could be detected by the system, we got a really long list of possibilities and picked 'capsaicin', which is responsible for the spiciness of edibles.
-First we looked for a sensor system which is able to detect substances of interest. Moreover we needed substances of interest.The system of interest has to be taken out of a differnent bacteria species than ''E.Coli'' , in order to avoid any background. Therefor we checked the literature for a well reviewed sensor system, which does not be part of the ''E.Coli'' genome. The system of choice is the phenolic sensing system virA of ''Aggrobacteria tumefaciens''. The systems naturally detects acetosyringone, which is a secondary metabolite of plants used as a bacterial attractant. So we hit the literature again to check for any other possible substances, which could be detected by the system. We got a really long list of possibilities and picked 'capcaicin', responsibily for spiciness of a fare.
+The biological steps for modulating the native VirA/G sensor system, in order to detect new substances are:
-The biological steps for creating a new sensor system are:
+# Extract the complete VirA/G signaling system out of ''A. tumefaciens''
+# Create new BioBricks out of the existing environmental parts
+# Get the new BioBricks working into ''E. coli''
+# Choose a reporter gene as readout for the sensor system
+# Modify the system for sensibility and specificity by error-prone PCR, find and select the most promising mutants (directed mutagenesis)
-# extracting the virA system out of ''A. tumefaciens''
+=== Preparing the system ===
-# create new biobricks out of the exciting environmental parts
-# transform the new biobricks into ''E.Coli''
-# modify the system for sensibility and specificity bt error prone pcr
-# find and select the most promising mutants
+We tried to work with the already existing VirA receptor BioBrick from the registry (<partinfo>K238008</partinfo>). Unfortunately this VirA BioBrick did not work in our experiments. We decided to sequence this BioBrick from the registry on our own and found that the actual sequence data of this BioBrick did not fit to the published sequence. Thus, in order to create this BioBrick we had to extract the ''virA'' gene via PCR out of the TI-plasmid from ''A. tumefaciens''.
-=== Preparing the system ===
+To construct the complete sensor system VirA is not enough, since it works only in combination with the VirG protein. The''virG'' gene was also present in the iGEM registry, but this version containes illegal restriction sites. Moreover it needs the help of the RpoA protein from ''A. tumefaciens'' to work as a transcription facor in ''E. coli''. In order to eliminate these drawbacks we changed the sequence manually and had the gene synthesized by Mr. Gene.
+=== Starting point for BioBricks -> Go cloning===
+After discussing the possibility of creating only one big plasmid for mutated ''virA'' screenings, we unified that its size would minimize transformation efficiency and would be difficult to modify via error-prone PCR. Thereby we decided to use a system with two plasmids per cell that offers certain advantages, which will be explained later. Prior to the construction of the plasmids we had to change the origin of a pSB1C3 plasmid in order to avoid incompatibility between both plasmids. Thus we cloned the R6K origin (<partinfo>J61001</partinfo>) into the <partinfo>pSB1C3</partinfo> plasmid and subsequently removed the original pMB1/ColE1 origin of replication. Therefore we digested the pSB1C3::R6K plasmid with Hin6I and seperated the resulting 6 fragments (1758 bp, 270 bp, 174 bp, 109 bp, 100 bp, 67 bp) by agarose gel electrophoresis. We extracted the largest fragment (1758 bp) and religated it. The resulting plasmid still contains 32 remaining basepairs of the pMB1 origin, but this fragment does not enable replication in pir<sup>-</sup> strains.
+[[Image:pSB1C3-R6K_300x300px.jpg]]  [[Image:pSB1C3-R6K-ColE1del_300x300px.jpg]]
+=== The error prone PCR and screening ===
+After building a plasmid with R6K ori we were able to create the two constructs. The first plasmid will be found inside the competent bacteria cells and contains the ''virG'' gene under the constitutive promoter <partinfo>J23110</partinfo>, a terminator (<partinfo>B0017</partinfo>), a ''vir'' promoter and a readout or selection gene (luciferase, mRFP and kanamycin resistance, respectively):
+[[Image:Screening_plasmid.jpg]]
+The second plasmid contains variants of ''virA'' ,which have been altered randomly by error-prone PCR, under the control of the constitutive promoter <partinfo>J23110</partinfo>.
+[[Image:VirA plasmid.jpg]]
+The error-prone PCR is a PCR using in inaccurate Taq polymerase under intesified malfunction conditions. It is possible to regulate the frequency of mutagenic events by using unbalanced concentrations of nucleotides and special salt concentrations in the PCR mixture. Using this mutagenesis we are going to create a new variants of the ''virA'' BioBrick and transform it in our cells with the read out system already present.
+=== Survival of the fittest ===
+We used high amounts of the antibiotic kanamycin in order to select the most specific systems for the detection of the chosen substances. By varying the amount of kanamycin we will be able to carefully select some promising clones out of thousands bacteria. The mutated ''virA'' system will be induced after the error-prone PCR by a mix of possible targets for the system (like capcaicin, dopamin, homovanillic acid etc.).
+=== Side directed mutagenesis ===
+Alternatively to the random mutagenesis we developed two in-silico based strategies for side directed mutagenesis as described by [http://www.nature.com/nature/journal/v423/n6936/abs/nature01556.html Looger ''et al.''].
+'''VirA_mut1'''
+to develop a strategy for change in the protein sequence, we used the analysis tool Muscle, which provided an advice to change one amino acid in the receptor. This conversion of leucin 293 to tyrosine was performed using site directed mutagenesis
+'''VirA_mut2'''
+Since VirA_mut1 with its single mutation failed working in our later screening system, we decided to mutate more amino acids in the area of the predicted binding region from animal TRPV1 receptors.
+According to the literature the amino acid residues 288 to 293 of this TRPV1 receptor regulate phenol structural specificity. Substitutions in this region narrow the active phenols to those missing the para-acyl substituent ([http://www.springerlink.com/content/978-0-387-78884-5#section=169995&page=1 Lin ''et al.'', 2008]). Our VirA_mut2 construct contains the following substitutions:
+*A357H *R358P *R359T *L360L *D361K *Y361L
+[[Image:Bielefeld_VirA_mut2.jpg|300px|thumb|center|'''Alignment of crucial ligand binding regions from ''Agrobacterium tumefaciens'' (lane 1), chick (lane 2), ''homo sapiens'' (lane 3), ''rattus'' (lane 4) and ''mus muskulus'' (lane 5). The Box shows all six amino acids we mutagenized in our VirA_mut2 approach''']]
+=== The final construct===
+In order to get a visible light signal by the reporter gene, we integrated the quantifiable proteins luciferase and mRFP. We also experimented with certain sensitivity tuners to be able to enhance the detected signal.
-We tried to work with the already existing virA out of the registry. Unfortunately this virA Biobrick did not work at all. The sequenced data of the biobrick did not fit to the published virA sequence. So we had to extract the virA gene via PCR out of the ''A. tumefaciens'' genome by ourselves in order to create a new biobrick.
-VirA does not work without the help of the VirG protein. The existing virG gene in the iGEM registry contains illegal restrictions site. Moreover it need the help of Chev Protein and sugars to work perfectly with virA. We decided to get some work done by MR. Gene. So we change the sequence manuelly and get the gene synthesized.
-==== Creating new biobricks ====
-The applied system in ''E.Coli'' consists of two plasmids. After discussion the possibility of creating only one big plasmid, we unify that one plasmid will minimized the transformation chance and will be difficult to modify via error prone pcr. So we had to change the origin of the psb1C3 plasmid in order to avoid compatibility problems at the transformation. Therefor we cloned the r6K origin into the psb1C3 plasmid. We used the r6k sequence and the psb1c3 sequence to create primers for the pcr following the ligation of the pcr products.
+For a demonstration of this final system see the animation below:
-After cloning the new origin into the psb1-backbone we are able to create the two constructs. The first one will be inside the competent bacteria cells and contains the
+[[Image:Bielefeld_Vorgehen.gif]]

Team:Bielefeld-Germany/Project/Approach