Hrp Characterisation: Notes
23rd of July
Meeting With the Hrp Research Group
- We arranged a meeting with a member of the Hrp lab to discuss our proposed methods for characterisation of the Hrp system.
- We initially began by explaining our ideas of how we can characterize the Hrp system. Afterwards, we discussed in greater details some of the aspects of the characterisation that the research group may have information on.
- The first question was about the types of promoters that would give a tightly regulated promoter. The PBAD promoter was suggested to us, this promoter is induced by addition of arabinose and repressed by araC. However, although this is a tightly regulated promoter it still had leaky expression. We were advised to add a high concentration of glucose in order to minimize this leakiness. In addition a paper  was recommended to us and we were told that we would be able to use the PBAD vectors that the lab had in stock.
- We also asked about any specific growing conditions for optimal Hrp activity. A minimal medium with an additional nutrient growth was advised to us.
- The next questions concerned specific features we wished to investigated:
- We asked about the possibility of using the Hrp in a variety of hosts cells. The only problem with this may be that if we decide to use gram+ and gram- hosts different vectors may be required.
- We then asked about the different temperatures that the Hrp system could work under. There research identified temperatures of about 25o were optimal. This is because the Hrp system would naturally be working in plants that are exposed to temperatures of around 25o.
- We discussed the idea of measuring PoPS, and asked about the feasibility of our proposal. after explaining it there seemed to be no immediate problems with what we had proposed.
- The meeting was a good step in trying to develop a protocol to characterize the Hrp, again the research group has been very helpful and has helped guide us to useful information.
- Guzman LM, Belin D, Carson MJ, and Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121-30. DOI:10.1128/jb.177.14.4121-4130.1995 |
Proposal for Lab
The first stages of this characterisation will be the preparation of our vectors and E.coli cultures, this will involve a series of cloning and transformation experiments. The techniques used will be the same as the other groups, the only difference being in the genes and vectors used. The strains that we plan to use will be E.coli K12 derivatives. The scecond stages of the characteristion is to use a fluorometer to measure the flourescence of a reporter gene under the control of inucible promoters. For this we require to grow our E.coli and then expose them to various concentrations of inducer. Various time points and inducer concentrations will be used to gather the data. The final experiments used is to carry out a series of dilution plate experiments on the E.coli used for the experiments mentioned above. The techniques we require do not prevent any safety issues, they use standard labatory materials and equipment.