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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

• miniprep of virA in the uc vector, fragment was digested a the gelelectrophoresis showed a good result
• new restriction digest for virB was done
• TOPO-cloning for rpoA failed again, so due to deadlines we decided that there was to few time left to do the TOPO-cloning again or with new protocols and to do the mutagenesis also.

• Because virA was now properly inserted in the pucvector we could now start mutagenesis on restriction sites 187 and 720. We did this with PCR. We used a protocol for site-directed-mutagenesis with the matching primers. PCR was done overnight
• new ligation for virB was done

• Gel-electrophoresis showed bad result for the mutation of virA probably due to too long elongation time. So we performed this PCR again with a new mutation.
• Ligation of virB didn't showed the expected result so we did it again.

• New PCR showed a better result for virA and geleclectrophoresis showed some result. The motherstrain was cut with DpnI to remove those residues, PCR-purification was performed and a restriction digest with PstI was done to see if restriction sites indeed were mutated
• virB ligation in K was again

• We checked if the PstI had cut the mutagenated places by gel-electrophoresis but we forgot to add a sample as positive control so the restriction digest was done again. The result we put on gel and saw that there was no cutting in both samples. Restriction disgest with pstI was now done overnight.
• virB ligation showed good results so it was electroporated