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 Knowledge on the HRP system

• HrpR and HrpS has optimal activation when both are expressed together in an operon. Transcription of this operon in a continuous polycistronic mRNA, that is then translated separately into HrpR and HrpS proteins.
• The HrpV repressor protein binds to the HTH motif of HrpS. The HrpV binds to the central region of HrpS, however its mechanism of binding is unknown.
• The DNA sequence within the HrpL promoter 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.
• The Hrp System when in Pseudomonas has an optimum temperature of28^oC.
• Three other chassie considerations is that Lon proteins have been shown to degrade HrpR, sigma 54 is required and Integration Host Factor is required.
• 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.
• Repression by HrpV reduces the original activity of the HrpL promoter to 5-10%. For full repression of the activity, it has been shown that large amounts of HrpV should be expressed before expressing HrpR and HrpS.