Team Plant Promoter consisted of Ryan and Niall.
Their objective was to amplify and generate three level 0 constructs containing plant promoters taken from Arabidopsis thaliana . In addition these promotors were then used to and generate level 1 phytobricks containing either luciferase or the TSH antagonist.
We isolated the plant promoters from Arabidopsis thaliana genomic DNA before amplifying the promoter sequences using PCR with primers that would incorporate BsmB1 type IIS restriction endonuclease recognition site. The amplfied DNA was then introduced into level 0 plasmids. This is documented on the basic parts page. We chose to only amplify the 3'-most 500bp of promoter DNA in order to facilitate future level 1 cloning reactions.
These promoters are PDF1.2 (jasmonic acid inducible), PR2 and GST6 (salicylic acid inducible).
These were then used to create level 1 constructs containing Luc+ or our TSH antagonist with 'His'-tags.We successfully generated level 1 constructs containing the TSH-antagonist Agrobacterium with PDF1.2, PR2, and GST6 were produced, and can be seen on our composite parts page. Unfortunately, the promoter:luc+:NosT constructs grew in E. coli , but not in Agrobacterium when we ran out of lab time, so these constructs could not be tested.
We transformed Nicotiana benthamiana plants with these successful level 1 contructs and performed protein extractions from leaves that had been induced with the appropriate plant hormone. Although the resulting coomassie stained gels did not contain any clear bands that correspond to the TSH-His (as crudely purified using nickel beads) we are hopeful that this protocol can be further optimised in future work. This might involve purifing the TSH-His protein with using different concentrations of nickel beads, altering the plant hormone induction protocol or using the more sensitive silver staining to identify unique bands. These gels are included on our results page.