IGEM:IMPERIAL/2007/Projects/Hrp System

Hrp Characterisation: Introduction

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Summary

The Aim of this project is to contribute new characterised parts to the biobricks registry. The parts we plan to contribute are from the Hrp system. The Hrp system is composed of a promoter under the control of two activating proteins and one repressing protein. The Hrp system can be manipulated in a number of ways to give various devices:

• A two input AND gate
• A three input AND gate with one inverted input

Our aim is to carry out a detailed characterisation of the Hrp parts and the various devices. We feel that this is going to be an important contribution to the biobricks registry for the following reasons:

• The Hrp system appears to have very interesting properties, being a highly regulated and responsive system, we feel that it will be a key addition to the registry and future iGEM projects.
• Our aim is to characterise Hrp thoroughly, we wish to make our parts and devices as generic and easy to use as possible. We feel that thorough characterisation is of great importance to the registry and to synthetic biology.We hope to illustrate this importance and to inspire current and future parts to have this level of characterisation.

The Hrp System

In Pseudomonas syringae strains, the hrp-hrc pathogenicity island consists of an HrpL-dependent regulon that encodes a type III protein translocation complex and translocated effector proteins required for pathogenesis. HrpR and HrpS function as positive regulatory factors for the hrpL promoter, but their mechanism of action has not been established. Both HrpR and HrpS are structurally related to enhancer-binding proteins, but they lack receiver domains and do not appear to require a cognate protein kinase for activity.

Starting from the current biological knowledge gathered on the Hrp System, this project intends to explore the potential behind this genetic circuit as a synthetic biology device. What would be its function ? How to characterize it ? How modular and generic can it be ?

To know more, our project idea proposal can be found here.

PPT

Current Information on the HRP system

• HrpR and HrpS functions most optimally when both are expressed together in an operon in a continuous manner
• Transcription of HrpR and HrpS results in a continuous polycistronic mRNA. They are then translated separately from a single mRNA.
• HrpV binds to the HTH motif of HrpS, not HrpR. HrpV binds to the central region of HrpS, but how it binds is still not known.
• Lon protein can be used to degrade HrpR.
• In Pseudomonas, the optimal temperature for operation of the system is 28oC; in the E.coli vector, the temperature used can range from 20-37oC. 25oC is the default temperature to work with.
• RT-PCR can be used to detect the level of transcription, but it takes time and only gives an estimate of the amount of mRNA, instead of the amount of proteins.
• The DNA sequence that HrpR and HrpS bind is unknown. What is known is that it is located somewhere upstream of the +1 position. Tests have shown that it must be within a 150bp range from the +1 site of transcription.

Some problem areas to solve

• HrpR and HrpS are constitutively active. HrpV is constitutively produced as well.
• HrpV negative regulation reduces the original activity of the system to 5-10%. For full repression of the activity, we need to express a large amount of HrpV first before expressing HrpR and HrpS. Another method would be to use the Lon protease to degrade HrpR.
• The half life of HrpR and HrpS is unknown.
• Inducers used for this system are arabinose and IPTG. If we use arabinose, a high concentration of glucose would be required to completely shut down the promoter of HrpR and HrpS.
• HrpL is Pseudomonas specific, and cannot operate in E.coli.
• HrpR and HrpS will can induce the promoter independent of each other. However, this is only about 2.5% activation compared to when both are present.
• Need to clone 300bp upstream of the HrpL promoter to ensure we have included the HrpR and HrpS binding site.
• Need to use a strain that contains the 'Integration Host Factor' IHF; this may limit our host choice.

Characterisation Tasks

• Write down quantitative specifications
• Find literature and protocols for each question
• Design experiments for each question
  • Note down the time requirements
• Plan the schedule