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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

• TOPO vector with insert W showed some colonies. PCR + electrophoresis were performed for control but a bad and unclear picture was made so the result was not that visible
• B and G vector were digested to perform gelextraction
• B and G were put on a gel ==> length of the fragments is OK
• Enting of R202 in fluid medium en tomorrow second mutation (427)

• PCR mutagenesis from R on 427, PCR purification and gelelectrophoresis: no signal on the picture probably because enting didn't show satisfying results
• Ligation van X and Y in K14 at 16°C
• New enting of R202
• W has been ented on solid agar, so miniprep and nanodrop are scheduled for today
• Mutagenesis of W + PCR purification

• PCR for the second mutation of R was done
• Electroporation of B and G after the ligation yesterday
• PCR was done for controle of W to check if virA was inserted into the cell. The picture made after gelelectrophorsis showed a wrong result

• Because of bad results yesterday the transformation of W was extra checked by performing a new PCR with different primers :

sample A : M13forward - virAforward sample B : M13forward - virAreverse sample C : virAforward - virAreverse sample D : M13reverse - virAforward sample E : M13reverse - virAreverse

Afterwards was gelelectrophoresis done and this showed virA was not present in the cell.

• ligation of X and Y showed a pover result so new ligations were done for virB and virG

• new TOPO-cloning was done but with a change in protocol. Ligationtime of the fragement in the vector was changed from 5 minutes to overnight.
• mutagenesis of rpoA on 202 was done but this showed no result on gel.
• A new PCR was done to have more virA

• TOPO cloning showed only blue colonies so we searched for alternatives. We did resaerchwork if it was maybe possible with a puc-vector
• The mutation of rpoA was again checked but gelelectrophoresis showed no result
• X,Y and K (the igemvector) were cut with EcoR1 and Pst1.
• new ligation was done with the first cuttingproduct so today it was electroporated and plated.
• PCR was done for W to get more basicproduct to work with

• There was a gel made to check if basicproduct W was correctly PCRed, and if the restriction digest of X,Y and K was good.
• rpoA mutation on 202 was put on gel but there was no result
• a plate was made of kolonies of Y+K ligation with aim to make a motherplate

• there was made a controllgel of virA for our basicproduct, purification and nanodrop
• the two ligations X+K were checked and electroporated
• X was digested again and ligated in K
• There were fluid cultures made for ligation of Y+K
• A new PCR was done for rpoA mutagenesis : elongationstep was now taken 4 minutes instead of 1 minute

• The Y+K ligation was miniprepped and nanodropped to make later on a stock on glycerol
• We decided to use for virA a puc19-vector in stead of TOPO, puc was digested with Xba1 and Ecor1 and virA was digested with EcoR1 and Spe1. This was done overnight.
• electroporation + plating was done for X+K

• gelextraction and nanodrop for puc and A. The digest was done well.
• mutagenesis was tried with colony pCR on position 428

• a new restrictiondigest was done for puc and A because we decided that earlier results were not good enough.
• There was a gelextraction, nanodrop and ligation done overnight.
• The colony PCR of rpoA => 428 again for one minute => bad result
• Miniprep of X+K was checked and put on gel. Wrong result probably because we selected a wrong colony.

• restriction digest of A and puc were done
• overnight ligation of puc and A

• fluid cultures were made of rpoA, X+K and the motherplate of virG so we could miniprep this tomorrow
• ligation of puc and A was electroporated
• new ligation of X+K was tried out

• puc+A was plated
• X+K was again elctroporated

• Glycerolstock was made for virG and a sample was send for seqencing
• virA was ented
• new TOPOcloning was done for rpoA with a new protocol we recieved