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• Check biobricks for vanillin detection

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

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

• [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.
  

• VirA

  VirG

  RpoA

  VirB promoter region

• 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.

• There was no result from our setup on Friday. We think there was an error with the protocol because we didn't shake before incubation. So we did a new transformation of every gene.

• We also received another strain of Agrobacterium. We brought this new strain in culture.
• To make things clear the genes from the first Agrobacterium will from now on be called A,B,G and R for respectively virA, promotor region virB , virG and rpoA. For the second Agrobacterium we will use W,X,Y and Z to indicate the same genes.
• We achieved good results for genes B,G and R. There were clear white colonies after plating the competent cells with the TOPO vector on X-gal agar plates with ampicillin. We isolated the good colonies again.
• There was no result for A. This was probably due to a fault with the competent cells. So we started TOPO transformation by heatshock all over for A.

• B,G and R were stored for further procedure. A failed again: there was no visible growth. This second time there was no problem with the competent cells because we used a commercial product instead of a self made. We assumed there was a problem with adding the polyA-tails to the PCR product because, when redoing the gelelectrophoresis, there were no errors with the PCR product.
• We did a new PCR on the bacteria with the inserted plasmids for B,G and R to check if the gene was inserted correctly.
• PCR was performed for W,X,Y and Z to isolate the genes.

1. B and R miniprepped. Tomorrow proceed with mutagenesis of RpoA
2. G redone (only TOPO was visible, 200bp) PCR from 2,3 and 1'
3. W, X, Y, Z PCR purification to create a TOPO vector tomorrow

Nanodrop:

1. G 2,3,1' on gel electrophoresis
   • 2 and 3 seem oke, 1000bp
   • 1' seems oke
   • 800bp fragment + 200bp TOPO)
2. Inoculate 2 and 3 and grow on ....
3. TOPO from W and Z and again from A
   • Note: if A fails again (with the iGEM protocol) then proceed with a new PCR purification.
4. Mutagenesis 1 on position 202 of R was performed

• A failed again so we decided to work only with W instead of performing a new PCR. TOPO for W and Z worked because there were colonies visible. White colonies were plated out.
• Fluid cultures of G2 and G3 were miniprepped, nanodropped and cut with Dpn I to exclude the motherstrain
• Mutagenesis 2 on R on position 427 was performed with PCR

• PCR purification miniprep and nanodrop of R on mutation 2
• Start ethanol precipitation (overnight ethanol + sodium acetate) to create a better result (more purified product) of product R with mutation 202
• PCR of the fragments of TOPO (W and Z) was done to check if TOPO worked. Gelelectrophoresis showed nothing so TOPO cloning failed again for virA.

• Continued with ethanol precipitation for R. Electroporation and outplating were performed

• Restriction digest of X, Y and with EcoR1 and Pst1
• Gelelectrophoresis to check fragments and gelextraction
• Fluid medium from outplated cells (overgrowth)
• TOPO-cloning (with new protocol from Invitrogen) for W and Z

• TOPO cloning of W looks goods with clear white colonies. 5 colonies were selected and cultured for testing