Team:IIT Delhi 1

 

Renin Angiotensin Aldosterone System (RAAS)

 

Introduction

The Renin Angiotensin Aldosterone System is a hormone system in the human body that is responsible for the regulation of blood pressure and fluid balance.

Functioning

A decrease in renal perfusion causes the release of the enzyme renin which subsequently cleaves angiotensinogen to angiotensin I. This is further converted to angiotensin II by angiotensin-converting enzyme (ACE). The bioactive product, angiotensin II, causes the constriction of blood vessels, leading to an increased blood pressure. It is also responsible for the secretion of aldosterone by the adrenal cortex, which acts by increasing the re-absorption of sodium and water into the blood. Hence, the fluid volume as well as the blood pressure is maintained in the body.

The α-hemolysin System in E.coli

 

Introduction

The alpha-hemolysin system is a type I secretion system found in wild type E.coli which enable them to cause infection in the urinary tract and is an important virulence factor because of its cytotoxic and cytolytic activity against a variety of mammalian cells.

Functioning

The hemolysin system originates from a series of hly genes. These genes are present in the series hlyC – hlyA – hlyB – hlyD. Of these, hlyA codes for the protein that is responsible for the infection. Hly B and D are membrane proteins that work in coordination for the secretion of HlyA. The synthesis, activation and secretion of HlyA in E.coli is determined by the hlyCABD operon. This operon is either located either on the chromosome or on transmissible plasmids.

It has been shown that this system can be used to produce and secrete a protein of our choice if the protein contains 60 amino acid residues from the carboxy terminal end of the HlyA protein.

Our iGEM 2010 Project

 

Introduction

Our aim is to create an online elicitor detection and response in a flow system, which can function in the detection of high blood pressure and help reduce it by releasing renin inhibitors into the circulatory system. This kind of a system would be helpful to patients with a hypertensive condition.

Approach

Since the blood pressure system is not really a chemical signal, what we have assumed is that the blood pressure can be easily converted into a chemical signal. Having assumed this, we will use a chemical signal such as IPTG or AHL as the elicitor to produce appropriate amounts of renin inhibitor. For easy detection and verification, we are planning to use GFP in place of renin inhibitor which can easily be replaced later.

For this we will be introducing two vectors into E. coli as shown below.

 

The first vector which contains the GFP-hlyA chimeric sequence with pLac promoter is used to produce the chimera GFP protein which contains 62 amino acid residues from the C terminal of hlyA protein, a prerequisite for the secretion of the protein outside the cell body. Forming a chimera will not in any way change the structure or function as per literature.

 

The other vector contains the hlyB and hlyD sequences with ptet promoter sequence. These two proteins will be constitutively expressed. These proteins are present in the membrane and provide for an outlet of hlyA or chimeras with 62 amino acid residues of the C terminal of hlyA.

After we are successful in obtaining GFP-hlyA chimera outside the cell body, our next task will be to immobilize these cells in a flow system and characterize the flow-expression profile. We would want to keep the cells in G0 phase so as to have continuous expression of the protein without cell death

We will also be working towards minimizing the toxins and other products so that such a system can finally be thought fit for incorporating and integrating with the circulatory system in our body.