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• Check biobricks for vanillin detection

• Mail University of Valencia because of inconsistent sequence in the parts registry [Done]

Received mail from Valencia: The vanillin Receptor is not usable, Madrid also tried to use and ran into all sorts of problems: Problems span from the binding of the periplasmic protein to the vanillin molecule, to the function of the fusion protein, to the activation of the ompR.

There are two solutions to this problem:

1. Construct a different vanillin receptor
   • Using virA/virG, a phenol receptor in agrobacterium .
2. Detect an alternative substance that has a linear relationship with the vanillin concentration
   • Vanillin inhibits the Ahl receptor
   • Synthesis of another signaling molecule that will be sensed

Possible receptor is the virA/virG system from Agrobacterium, the virA protein will bind to phenolic compounds and induce transcription of vir genes by phosphorylation of virG. Protein ChvE results in a broader response to phenolic compounds. Papers:

• [1] S C Winans, Two-way chemical signaling in Agrobacterium-plant interactions, Microbiol Mol Biol Rev. 1992 March; 56(1): 12-31
  
• [2] Wen-Tao Peng, Yong-Woog Lee, and Eugene W. Nester, The Phenolic Recognition Profiles of the Agrobacterium tumefaciens VirA Protein Are Broadened by a High Level of the Sugar Binding Protein ChvE, Journal of Bacteriology, November 1998, p. 5632-5638, Vol. 180, No. 21
  
• [3]Yong-Woog Lee, Shouguang Jin, Woong-Seop Sim, Eugene W. Nester, The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA protein, Gene, Volume 179, Issue 1, 1996, Pages 83-88, ISSN 0378-1119, DOI: 10.1016/S0378-1119(96)00328-9.
  
• [4]Y W Lee, S Jin, W S Sim, and E W Nester,Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens, Proc Natl Acad Sci U S A. 1995 December 19; 92(26): 12245–12249.
  

Template:Team:KULeuven/27 July 2009/VanillinReceptor

The primers for the following components of the receptor were ordered:

• VirA

  VirG

  RpoA

  VirB promoter region

Template:Team:KULeuven/29 July 2009/VanillinReceptor

Template:Team:KULeuven/30 July 2009/VanillinReceptor

The primers for site directed mutagenesis were made:

• Two primer-sets for mutating the PstI site out of VirA
• Two primer-sets for mutating the EcoRI site out of RpoA

• Agrobacterium tumefaciens strain pTiA6NC/A1011 was plated out on LB-medium.
• Primers for virA, virG, promotor region virB and RpoA have arrived

• The primers made for the directed mutagenesis on Friday were ordered
• PCR was done with primers for virA, virG, promotorregion virB and RpoA with template DNA from Agrobacterium tumefaciens pTiA6NC/A1011 to isolate the genes

• Gel electrophoresis was done to see if PCR has worked. VirA, virB and rpoA showed a clear result. We performed miniprep on those genes to purify and clear them. VirG didn't show any result.
• We did a new PCR for all the genes and especially for virG with a better annealing temperature. The gel electrophoresis showed a good and clear result for all the genes.

• VirA, virB, virG and rpoA were purified and nanodropped
• All the genes were elongated with polyA-tails to make it possible to insert them into a TOPO-vector

• Primers for mutagenesis have arrived
• We used the DNA parts from the second PCR to insert them into a TOPO-vector. The second PCR showed more results so that is why we used these fragments.
• LBmedium was made with X-gal to select cells afterwards.
• Plasmids were inserted in competent cells by heatshock and those cells were plated out on the X-gal medium.