Renin Angiotensin Aldosterone System (RAAS)<o:p></o:p></span></h2>
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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.
<h2>The α-hemolysin System in E.coli<o:p></o:p></h2>
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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.
<h2>Our iGEM 2010 Project<o:p></o:p></h2>
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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.
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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.
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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.
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