Vanillin Receptor: Planning
Besides producing vanillin, we would also like to control the concentration of vanillin outside of the bacteria. To do this, we need a way to detect the vanillin extracellular and create a proper response intracellular. The vanillin sensing system receptor we propose, consists of the VirA/G system normally found in the Agrobacterium Tumefaciens. VirA is a receptor that responses to monosaccharides and phenols. Hence, it is able to sense vanillin. When binding vanillin, VirA is activated and will activate in its turn VirG. VirG is a transcription activator that will bind to a Vir box sequence in order to activate gene expression. This Vir box sequence can be found in the VirB promoter region. RpoA is a alfa subunit polymerase that is needed to help VirG work in E. coli. For extra literature see: 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.
- Strains/isolated DNA of Agrobacterium tumefaciens: pTiBo542 and pTiA6NC/A1011
For VirA, VirG, RpoA, VirB promoter region and for the site directed mutagenesis to remove unwanted restriction sites in VirA (PstI 2x) and RpoA (EcoRI 2x)
- TOPO-Vector or iGEM vector
- The primers were ordered
- The plasmids: from the lab
- One strain was ordered and another was available in the lab
- The Agrobacterium strain is plated out on agar from -80°C. From the other strain, DNA is expected to be sent.
- The primers are used to perform a PCR on the bacteria or on the isolated DNA to amplify the different genes or fragments (VirA, VirG, RpoA and virB promoter region). An agarose is performed as a control.
- The PCR fragments are ligated in a TOPO vector and electroporated into E. coli.
- The plasmids are isolated via miniprep and again verified through restriction digest and agarose gel.
- Site directed mutagenesis (according to stratagene) is performed on RpoA and VirA to get the EcoRI and PstI restriction sites out.
- All parts are sequenced.
- The different plasmids (consideration: put in registry as separate part) are combined into one standard iGEM plasmid, with the appropriate promoter, RBS and terminators.
- The system’s function is checked by linking a GFP to the virB-promoter region and subsequent induction with vanillin and Ferulic Acid