IGEM:IMPERIAL/2007/Projects/Hrp System: Difference between revisions

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<li id="current">[[IGEM:IMPERIAL/2007/Projects/Hrp System|Introduction]]</li>
<li id="current">[[IGEM:IMPERIAL/2007/Projects/Hrp System|Introduction]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Specifications|Specifications]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Specifications|Specifications]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Design|Design]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Parts|Parts]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Modelling|Modelling]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Devices|Devices]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Implementation|Implementation]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Testing|Testing]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/TestingValidation|Testing/Validation]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Application|Application]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Notes|Notes]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/Notes|Notes]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/References|References]]</li>
<li>[[IGEM:IMPERIAL/2007/Projects/Hrp System/References|References]]</li>
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=== 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 positive regulating proteins and one negative regulating protein. The Hrp system can be manipulated in a number of ways to give various devices:
The Aim of this project is to contribute new well 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 new regulatory system
* A two input AND gate
* A two input AND gate
* A three input AND gate with one inverted input
* A three input AND with one input inverted  
Our aim is to carry out a detailed characterisation of the Hrp parts and the devices. We feel that this is going to be an important contribution to the biobricks registry for the following reasons:
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.
* 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.  
* 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.
We wish to characterise the Hrp system in generic units that allow modular design and experimental reproducibility. This is a key concept in synthetic biology, with the ideal unit being [[IGEM:IMPERIAL/2007/Projects/Hrp System/Notes/PoPS| Polymerase Per Second (PoPS)]], However, we cannot directly measure PoPS and so we haev to measure it indirectly. One approach is to measure rate of protein synthesis and relate this back to PoPS. We propose to measure our device in terms of protein synthesis, particularly '''GFP molecule synthesised cfu<sup>-1</sup> sec<sup>-1</sup>'''.
This measurement we feel is still a valid generic unit:
*It is a reproducible measurement because the unit is independent of the equipment used, such as fluorometers that can vary in calibrations. This independence is because we are measuring in terms of the rate of GFP synthesis as opposed to just fluorescence.
*The unit also allows modular design, this is either when the other parts used have the same units or the rate of GFP synthesis is related to a more generic unit for protein synthesis or to PoPS. We will be measuring the input and output of our Hrp system in terms of GFP synthesis.


=== Summary of 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.
== The Hrp System ==
 
[[IMAGE:ICGEMS_dvc_HrpR&S+V+L.png‎|thumb|right|230px| The Hrp System]]
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 ?
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.  
 
<br>
To know more, our project idea proposal can be found here.
HrpR and HrpS function as positive regulatory factors of the hrpL promoter, but their mechanism of action has not been established. HrpV is a negative regulatory factor of the hrpL promoter, its action will repress any activation by HrpR and HrpS.
 
<br>
[[Media:icgems_hrp_proposal.ppt|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.
===Why HRP system is optimized for biobricks===
*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.
*Residual activity of the system is very low, and only when the number of vectors are increased that noise can be detected.
*The Hrp System when in Pseudomonas has an optimum temperature of 28 <sup>o</sup>C.  
*PsPF does not compete with HspL unless it is overexpressed. Hence, the system does not suffer from cross talk and is isolated in E.coli.
*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 does not bind to any other sigmafactor other than sigma 54. Hence they are very specific.  
*The system can be switched on or off very quickly (when working with E.coli as a vector), as ATP is part of the regulation and can be phosphorylated and dephosphorylated in a matter of seconds.
*There is no leaky transcription.
*The system is very sensitive.
 
===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.
*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.
*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.
*Need to use a strain that contains the 'Integration Host Factor' IHF; this may limit our host choice.
 
===Some points to note about 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.
 
=== 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

Latest revision as of 02:37, 3 August 2007

Hrp Characterisation: Introduction


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 positive regulating proteins and one negative regulating protein. The Hrp system can be manipulated in a number of ways to give various devices:

  • A new regulatory system
  • A two input AND gate
  • A three input AND with one input inverted

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.

We wish to characterise the Hrp system in generic units that allow modular design and experimental reproducibility. This is a key concept in synthetic biology, with the ideal unit being Polymerase Per Second (PoPS), However, we cannot directly measure PoPS and so we haev to measure it indirectly. One approach is to measure rate of protein synthesis and relate this back to PoPS. We propose to measure our device in terms of protein synthesis, particularly GFP molecule synthesised cfu-1 sec-1. This measurement we feel is still a valid generic unit:

  • It is a reproducible measurement because the unit is independent of the equipment used, such as fluorometers that can vary in calibrations. This independence is because we are measuring in terms of the rate of GFP synthesis as opposed to just fluorescence.
  • The unit also allows modular design, this is either when the other parts used have the same units or the rate of GFP synthesis is related to a more generic unit for protein synthesis or to PoPS. We will be measuring the input and output of our Hrp system in terms of GFP synthesis.


The Hrp System

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 of the hrpL promoter, but their mechanism of action has not been established. HrpV is a negative regulatory factor of the hrpL promoter, its action will repress any activation by HrpR and HrpS.

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 of 28 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.
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