Team:Colombia Uniandes/Project

From 2013.igem.org

(Difference between revisions)
(Reporter for PrcnA)
 
(30 intermediate revisions not shown)
Line 1: Line 1:
{{Http://2013.igem.org/Team:Colombia Uniandes/Bootstrap}}
{{Http://2013.igem.org/Team:Colombia Uniandes/Bootstrap}}
-
== '''ColombiaSuperProjects!''' ==
+
<div class="container">
-
===Our Stress Sensor! Glucocorticoid based===
+
    <div class="span11">
 +
<center><h2>ColombiaSuperProjects!</h2></center>
 +
<div class = "span5">
 +
<div class="alert alert-info">
 +
<h3>Index</h3>
 +
<ul>
 +
<li>[[#Gluco|Our Stress Sensor! Glucocorticoid based]]</li>
 +
<li>[[#Nickel|Our Nickel Absorption System!]] </li>
 +
<li>[[#parts|Design: Project Parts!]] </li>
 +
<ul>
 +
<li>[[#glucoSensor|Glucocorticoid sensor]]</li>
 +
<ul>
 +
<li>[[#glucoConstruct|Our construct]]</li>
 +
<li>[[#glucoChassis|The Chassis]]</li>
 +
<li>[[#glucoChimera|The Chimera]]</li>
 +
<li>[[#glucoTester|Tester Construct]]</li>
 +
</ul>
 +
<li>[[#nickelRemoval|Nickel Removal System]]</li>
 +
<ul>
 +
<li>[[#nickelConstruct|Our Construct]]</li>
 +
<li>[[#nickelExplanation| Explanation?]]</li>
 +
<li>[[#nickelChassis|Our chassis]]</li>
 +
<li>[[#nickelReporter|Reporter for PrcnA]]</li>
 +
<li>[[#nickelProgress|Our work in progress!]]</li>
 +
</ul>
 +
</ul>
 +
 
 +
<html>
 +
</div>
 +
</div>
 +
 
 +
</html>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<h3 id="Gluco">Our Stress Sensor! Glucocorticoid based</h3>
[[File:Chimi.jpg|300px|thumb|left|'''Chimi''']]
[[File:Chimi.jpg|300px|thumb|left|'''Chimi''']]
Line 11: Line 73:
Our aim is to create a glucocorticoid sensor that is able to discern between basal levels and stress levels of glucocorticoid hormones in a sample with an easily recognizable signal, such as color, to allow the sensor to be used in the field, household or the laboratory.
Our aim is to create a glucocorticoid sensor that is able to discern between basal levels and stress levels of glucocorticoid hormones in a sample with an easily recognizable signal, such as color, to allow the sensor to be used in the field, household or the laboratory.
</p>
</p>
-
===Our Nickel Absorption System!===
+
<h3 id="Nickel">Our Nickel Absorption System!</h3>
[[File: Nicko.jpg|300px|thumb|right|'''Nicko''']]
[[File: Nicko.jpg|300px|thumb|right|'''Nicko''']]
Line 22: Line 84:
</p>
</p>
<p align="justify">
<p align="justify">
-
Responding to these concerns, we have designed a bacterium which would be capable of absorbing the nickel in its interior using the internal regulating system of nickel belonging to the ''Ralstonia metallidurans'', to later be removed magnetically, using the property from ''Magnetospirillum magneticum'' AMB-1 which will be used as our final chassis.
+
Responding to these concerns, we have designed a bacterium which would be capable of absorbing the nickel in its interior using the internal regulating system of nickel belonging to the ''Cupriavidus metallidurans'', to later be removed magnetically, using the property from ''Magnetospirillum magneticum'' AMB-1 which will be used as our final chassis.
</p>
</p>
-
=='''Design: Project Parts!'''==
+
<h3 id="parts">Design: Project Parts!</h3>
-
== Glucocorticoid sensor==
+
<blockquote>
-
===Our construct===
+
<h4 id="glucoSensor"> Glucocorticoid sensor</h4>
 +
<blockquote>
 +
<h5 id="glucoConstruct">Our construct</h5>
<p align="justify">
<p align="justify">
We plan to use the baker's yeast, ''Saccharomyces cerevisiae'', as a chassis for a plasmid which will contain a chimeric protein used as a transactivating factor in a biosensor with a colored reporter.
We plan to use the baker's yeast, ''Saccharomyces cerevisiae'', as a chassis for a plasmid which will contain a chimeric protein used as a transactivating factor in a biosensor with a colored reporter.
</p>
</p>
-
====The Chassis====
+
<h5 id="glucoChassis">The Chassis</h5>
<p align="justify">
<p align="justify">
We chose ''S. cerevisiae'' as the chassis because one of the most important parts of our fusion protein, the glucocorticoid receptor hormone binding domain (GCR HBD) is eukaryotic, therefore we wanted an easy to use, easy to grow, eucaryotic vector to express our protein and build our biosensor.
We chose ''S. cerevisiae'' as the chassis because one of the most important parts of our fusion protein, the glucocorticoid receptor hormone binding domain (GCR HBD) is eukaryotic, therefore we wanted an easy to use, easy to grow, eucaryotic vector to express our protein and build our biosensor.
</p>
</p>
-
====The Chimera====
+
<h5 id="glucoChimera">The Chimera</h5>
<p align="justify">
<p align="justify">
The glucocorticoid receptor (GCR) from mammals contains three domains necessary for stress hormone related gene transcription, the hormone binding domain (HBD), the DNA binding domain (DNA-BD) and the gene transactivating domain (GTD).
The glucocorticoid receptor (GCR) from mammals contains three domains necessary for stress hormone related gene transcription, the hormone binding domain (HBD), the DNA binding domain (DNA-BD) and the gene transactivating domain (GTD).
Line 45: Line 109:
We used the glucocorticoid receptor hormone binding domain (GCR HBD) which came from a rat to recognize our hormones of interest. However, we replaced the other two domains with the herpesvirus gene transactivating domain (HV-GTD) and the yeast's DNA binding domain from GAL4. These two new domains have the advantage of being already used, characterized and being highly efficient. The HV-GTD is a highly efficient transactivating domain, recognized to be several orders of magnitude better than the GCR-GTD.  
We used the glucocorticoid receptor hormone binding domain (GCR HBD) which came from a rat to recognize our hormones of interest. However, we replaced the other two domains with the herpesvirus gene transactivating domain (HV-GTD) and the yeast's DNA binding domain from GAL4. These two new domains have the advantage of being already used, characterized and being highly efficient. The HV-GTD is a highly efficient transactivating domain, recognized to be several orders of magnitude better than the GCR-GTD.  
</p>
</p>
-
===Tester Construct===
 
-
[[File:GCtestconstruct1.jpg | thumb | center | upright=3.0 | Glucocorticoid test construct]]
 
-
{| border="1" align="center"
+
</blockquote>
-
|+'''Individual Parts Checklist*'''
+
</blockquote>
-
| style="text-align: center;" |pBAP2
+
-
| style="background:green; text-align: center;"|''Extracted''
+
-
|-
+
-
| style="text-align: center;" |GAL4
+
-
| style="background:green; text-align: center;"|''Extracted''
+
-
|-
+
-
| style="text-align: center;" |VP16
+
-
| style="background:green; text-align: center;"|''Extracted''
+
-
|-
+
-
| style="text-align: center;" |GCR
+
-
| style="background:green; text-align: center;"|''Extracted''
+
-
|-
+
-
| style="text-align: center;" |Yeast Terminator
+
-
| style="background:blue; text-align: center;"|''In progress''
+
-
|-
+
-
| style="text-align: center;" |pGAL1
+
-
| style="background:green; text-align: center;"|''Extracted''
+
-
|-
+
-
| style="text-align: center;" |mCherry
+
-
| style="background:green; text-align: center;"|''Extracted''
+
-
|}
+
-
 
+
<blockquote>
-
{| border="1" align="center"
+
<h4 id="nickelRemoval">Nickel Removal System</h4>
-
|+'''Fusion Checklist*'''
+
<blockquote>
-
| style="text-align: center;" |pBAP2 + GAL4
+
<h5 id="nickelConstruct">Our construct</h5>
-
| style="background:green; text-align: center;"|''Fused''
+
-
|-
+
-
| style="text-align: center;" |VP16 + GCR
+
-
| style="background:green; text-align: center;"|''Fused''
+
-
|-
+
-
| style="text-align: center;" |VP16 + GCR + yeast Terminator
+
-
| style="background:red; text-align: center;"|''Stand by''
+
-
|-
+
-
| style="text-align: center;" |pBAP2 + GAL4 + VP16 + GCR + yeast Terminator
+
-
| style="background:red; text-align: center;" |''Stand by''
+
-
|-
+
-
| style="text-align: center;" |pGAL1 + mCherry
+
-
| style="background:green; text-align: center;"|''Fused''
+
-
|}
+
-
 
+
-
'''*'''Updated as of July 27.
+
-
 
+
-
==Nickel Removal System==
+
-
 
+
-
===Our construct===
+
<p align="justify">
<p align="justify">
-
Using the nickel homeostatic system of ''E. coli'' and ''Ralstonia metallidurans'', we will be able to create a system capable to detect and absorb nickel, to later be removed magnetically, using the magnetotactic property from ''Magnetospirillum magneticum'' AMB-1 which will be used as our final chassis.
+
Using the nickel homeostatic system of ''E. coli'' and ''Cupriavidus metallidurans'', we will be able to create a system capable to detect and absorb nickel, to later be removed magnetically, using the magnetotactic property from ''Magnetospirillum magneticum'' AMB-1 which will be used as our final chassis.
</p>
</p>
[[File:Img.jpg|700px|thumb|center|'''Nickel removal system construct''']]
[[File:Img.jpg|700px|thumb|center|'''Nickel removal system construct''']]
-
====Explanation?====
+
<h5 id="nickelExplanation">Explanation?</h5>
<p align="justify">
<p align="justify">
Prcnr is a constitutive promoter, constantly producing RcnR protein which represses PrcnA.  When there is nickel in the media RcnR alters its form, making it impossible to bind PrcnA, activating the production of the HoxN protein. This last protein is an efficient nickel transporter form ''Ralstonia metallidurans'' that places in the membrane allowing the income of the metal into the cell.  
Prcnr is a constitutive promoter, constantly producing RcnR protein which represses PrcnA.  When there is nickel in the media RcnR alters its form, making it impossible to bind PrcnA, activating the production of the HoxN protein. This last protein is an efficient nickel transporter form ''Ralstonia metallidurans'' that places in the membrane allowing the income of the metal into the cell.  
Line 110: Line 131:
</p>
</p>
-
====Our chassis====
+
<h5 id= "nickelChassis">Our chassis</h5>
<p align="justify">
<p align="justify">
We chose ''Magnetospirillum magneticum'' AMB-1 as a final chassis this organism has the particularity to produce “magnetosomes” which are vesicles that contain 15 to 20 magnetite crystals that together act like a compass needle to orient magnetotactic bacteria in geomagnetic fields. This property, will allow us to eliminate the bacteria from water with a simple magnetic field.
We chose ''Magnetospirillum magneticum'' AMB-1 as a final chassis this organism has the particularity to produce “magnetosomes” which are vesicles that contain 15 to 20 magnetite crystals that together act like a compass needle to orient magnetotactic bacteria in geomagnetic fields. This property, will allow us to eliminate the bacteria from water with a simple magnetic field.
</p>
</p>
-
====Reporter for PrcnA====
+
<h5 id="nickelReporter">Reporter for PrcnA</h5>
<p align="justify">
<p align="justify">
-
In order to check PrcnA obtained works in the presence of nickel, we will measure the RFP activity, using our construct ont only as a nickel removal system but as a quantitative nickel reporter as well.
+
In order to check PrcnA obtained works in the presence of nickel, we will measure the RFP activity, using our construct not only as a nickel removal system but as a quantitative nickel reporter as well.
</p>
</p>
-
====Our work in progress!====
+
<html>
-
 
+
</blockquote>
-
{| border="1" align="center"
+
</blockquote>
-
|+'''Individual Parts Checklist*'''
+
</div>
-
| style="text-align: center;" |PrcnR
+
</div>
-
| style="background:green; text-align: center;"|''Extracted''
+
</html>
-
|-
+
-
| style="text-align: center;" |rcnR
+
-
| style="background:green; text-align: center;"|''Extracted''
+
-
|-
+
-
| style="text-align: center;" |PrcnA
+
-
| style="background:blue; text-align: center;"|''In progress''
+
-
|-
+
-
| style="text-align: center;" |hoxN
+
-
| style="background:green; text-align: center;"|''Extracted''
+
-
|-
+
-
| style="text-align: center;" |GFP
+
-
| style="background:red; text-align: center;"|''Stand by''
+
-
|-
+
-
|}
+
-
 
+
-
* Updated July 15
+

Latest revision as of 22:41, 27 September 2013

Contents

ColombiaSuperProjects!
















Our Stress Sensor! Glucocorticoid based

Chimi

Glucocorticoids are hormones present in mammals that undergo changes in concentrations when the animal is under stressful stimuli. These hormones can be measured in blood plasma, urine, saliva and hair. Chemical methods are used to measure these hormones for veterinary and agricultural use, but these methods require expensive equipment and a capacitated technician which makes it uncomfortable and useless in the field.

Our aim is to create a glucocorticoid sensor that is able to discern between basal levels and stress levels of glucocorticoid hormones in a sample with an easily recognizable signal, such as color, to allow the sensor to be used in the field, household or the laboratory.

Our Nickel Absorption System!

Nicko

Nickel is silvery-white, hard, malleable, and ductile metal. Now days the major use of nickel is in the preparation of alloys which are characterized by strength, ductility, and resistance to corrosion and heat, such as stainless steel. Other uses are rechargeable batteries, catalysts and other chemicals among others. Most nickel on Earth is inaccessible; this is why the metal is mined in Russia, Australia, Cuba, Canada, Colombia, Venezuela and South Africa.

As a result of this activity all kinds of water bodies are being contaminated with metals of this nature, Colombia, is one of the many affected. In small quantities nickel is essential, but when the uptake is too high it can be a dangerous to human health bringing many types of cancer, birth defects, abortions, asthma, allergic reactions such as skin rashes or “nickel-itch”, and many other conditions as its consequences.

Responding to these concerns, we have designed a bacterium which would be capable of absorbing the nickel in its interior using the internal regulating system of nickel belonging to the Cupriavidus metallidurans, to later be removed magnetically, using the property from Magnetospirillum magneticum AMB-1 which will be used as our final chassis.

Design: Project Parts!

Glucocorticoid sensor

Our construct

We plan to use the baker's yeast, Saccharomyces cerevisiae, as a chassis for a plasmid which will contain a chimeric protein used as a transactivating factor in a biosensor with a colored reporter.

The Chassis

We chose S. cerevisiae as the chassis because one of the most important parts of our fusion protein, the glucocorticoid receptor hormone binding domain (GCR HBD) is eukaryotic, therefore we wanted an easy to use, easy to grow, eucaryotic vector to express our protein and build our biosensor.

The Chimera

The glucocorticoid receptor (GCR) from mammals contains three domains necessary for stress hormone related gene transcription, the hormone binding domain (HBD), the DNA binding domain (DNA-BD) and the gene transactivating domain (GTD).

However, for our construct's performance we used a chimeric protein. Just as the mythological creature made from fused parts from a lion, a goat and a snake, we created a chimeric protein using three domains from different organisms.

We used the glucocorticoid receptor hormone binding domain (GCR HBD) which came from a rat to recognize our hormones of interest. However, we replaced the other two domains with the herpesvirus gene transactivating domain (HV-GTD) and the yeast's DNA binding domain from GAL4. These two new domains have the advantage of being already used, characterized and being highly efficient. The HV-GTD is a highly efficient transactivating domain, recognized to be several orders of magnitude better than the GCR-GTD.

Nickel Removal System

Our construct

Using the nickel homeostatic system of E. coli and Cupriavidus metallidurans, we will be able to create a system capable to detect and absorb nickel, to later be removed magnetically, using the magnetotactic property from Magnetospirillum magneticum AMB-1 which will be used as our final chassis.

Nickel removal system construct
Explanation?

Prcnr is a constitutive promoter, constantly producing RcnR protein which represses PrcnA. When there is nickel in the media RcnR alters its form, making it impossible to bind PrcnA, activating the production of the HoxN protein. This last protein is an efficient nickel transporter form Ralstonia metallidurans that places in the membrane allowing the income of the metal into the cell.

Acording to the above, our system is recensusing for nickel everytime, and when this metal is detected, it is absorbed and consequently further increases its absorption.

Our chassis

We chose Magnetospirillum magneticum AMB-1 as a final chassis this organism has the particularity to produce “magnetosomes” which are vesicles that contain 15 to 20 magnetite crystals that together act like a compass needle to orient magnetotactic bacteria in geomagnetic fields. This property, will allow us to eliminate the bacteria from water with a simple magnetic field.

Reporter for PrcnA

In order to check PrcnA obtained works in the presence of nickel, we will measure the RFP activity, using our construct not only as a nickel removal system but as a quantitative nickel reporter as well.

Retrieved from "http://2013.igem.org/Team:Colombia_Uniandes/Project"