Team:Gothenburg/Project

From 2014.igem.org

(Difference between revisions)
m (Fixing)
(Styling and fixes)
Line 19: Line 19:
}
}
 +
.imgRight {
 +
float: right;
 +
}
</style>
</style>
Line 25: Line 28:
<body>
<body>
<div><!--main content -->
<div><!--main content -->
-
<table width="70%" align="center">
 
 +
<!--welcome box
 +
<tr>
 +
<td style="border:1px solid black;" colspan="3" align="center" height="150px" bgColor=#FF404B>
 +
<h1 >Welcome to the Wiki of Gothenburgs team for iGEM 2014! </h1>
 +
<p>This whole page is under very experimental construction.
 +
<br>In a not too distant future you will find all the amazing things we learned and achieved here. </p>
 +
<br>
 +
<p style="color:#F0F0F0"> <a href="https://2014.igem.org/wiki/index.php?title=Team:Gothenburg/Project&action=edit"style="color:#FFFFFF"> Click here to edit the project page!</a> </p>
 +
</td>
 +
</tr>
-
<!--welcome box -->
+
<tr> <td colspan="3" height="5px"> </td></tr>
-
<tr>
+
end welcome box -->  
-
<td style="border:1px solid black;" colspan="3" align="center" height="150px" bgColor=#FF404B>
+
-
<h1 >Welcome to the Wiki of Gothenburgs team for iGEM 2014! </h1>
+
-
<p>This whole page is under very experimental construction.
+
-
<br>In a not too distant future you will find all the amazing things we learned and achieved here. </p>
+
-
<br>
+
-
<p style="color:#F0F0F0"> <a href="https://2014.igem.org/wiki/index.php?title=Team:Gothenburg/Project&action=edit"style="color:#FFFFFF"> Click here to edit the project page!</a> </p>
+
-
</td>
+
-
</tr>
+
-
<tr> <td colspan="3"  height="5px"> </td></tr>
+
<!-- <tr> <td colspan="3"  height="15px"> </td></tr>
-
<!-- end welcome box -->  
+
<tr> <td bgColor="#e7e7e7" colspan="3" height="1px"> </tr>
 +
<tr> <td colspan="3"  height="5px"> </td></tr> -->
-
<tr> <td colspan="3"  height="15px"> </td></tr>
 
-
<tr> <td bgColor="#e7e7e7" colspan="3" height="1px"> </tr>
 
-
<tr> <td colspan="3"  height="5px"> </td></tr>
 
-
 
+
<!--Project content  -->
-
<!--Project content  -->
+
<h3> Project Description </h3>
-
<tr>
+
<p>
-
<td>
+
Nowadays the determination of replicative age of yeast cells is done by  
-
<h3> Project Description </h3>
+
counting the budding scars of each cell in a microscope, a process time consuming
-
<p>
+
and not effective. Our team goal with the iGEM project is to construct a yeast
-
Nowadays the determination of replicative age of yeast cells is done by  
+
generation counter. The idea is that each time the cell divides a different
-
counting the budding scars of each cell in a microscope, a process time consuming
+
florescent protein [1, 2] is produced. Therefore by examining the cell under a
-
and not effective. Our team goal with the iGEM project is to construct a yeast
+
microscope or in a flow cytometer one can determine how many times the cell has
-
generation counter. The idea is that each time the cell divides a different
+
divided. This would be achieved by constructing a logical AND gate in the cell [3]
-
florescent protein [1, 2] is produced. Therefore by examining the cell under a
+
where the input signals consist of a cyclin activated dCas9-VP64 and a guide RNA
-
microscope or in a flow cytometer one can determine how many times the cell has
+
(gRNA) [4] signal from the previous cell cycle. The output response consist of a
-
divided. This would be achieved by constructing a logical AND gate in the cell [3]
+
different fluorescent protein and a new gRNA molecule, see figure 1.
-
where the input signals consist of a cyclin activated dCas9-VP64 and a guide RNA
+
</p>
-
(gRNA) [4] signal from the previous cell cycle. The output response consist of a
+
<div class="imgRight">
-
different fluorescent protein and a new gRNA molecule, see figure 1.
+
<img src="https://static.igem.org/mediawiki/2014/8/88/Team_Gothenburg_And_Gate.png"  
-
</p>
+
width="300px" height="100px" />
-
<img src="" width="300px" height="100px" />
+
<br>
-
<p>
+
<p>Figure 1. Schematic representation of the logical AND gate with the input
-
dCas9-VP64 is an engineered turntable transcription factor only active when dimerized
+
and output signals.
-
with an interchangeable gRNA molecule. The gRNA also determines the specificity of  
+
-
the transcription factor which enables the dCas9-VP64 to activate different genes
+
-
depending on the sequence of the gRNA molecule and the promoter. gRNA consists of two
+
-
parts, a scaffold and a 20 bp Specificity Determinant Sequence (SDS) on the 5' end.
+
-
The scaffold constitutes the majority of the gRNA molecule and gives it its structure
+
-
whereas the SDS binds to the target site in the gene promoter.  Cyclins are proteins
+
-
that are involved in the progression of the cell cycle, therefore activated at specific
+
-
times [5]. To mimic the specific production pattern of cyclins the dCas9-VP64 gene is
+
-
placed under the control of a yeast cyclin promoter. Therefore the Cas9 will be
+
-
produced in the G1 phase. When the Cas9 and gRNA dimerize and the transcription factor
+
-
is activated, it in turn activates the transcription of a new fluorescent protein and
+
-
a new gRNA molecule to act as a memory for the next cycle. Once this age counter is
+
-
implemented, it would be possible to sort cells according to their replicative age
+
-
automatically with a flow cytometer device.
+
</p>
</p>
-
<h4>References</h4>
+
</div>
-
<ol id="references">
+
<p>
-
<li>Hackett, E.A., et al., A family of destabilized cyan fluorescent proteins as  
+
dCas9-VP64 is an engineered turntable transcription factor only active when dimerized
-
transcriptional reporters in S. cerevisiae. Yeast, 2006. 23(5): p. 333-349.</li>
+
with an interchangeable gRNA molecule. The gRNA also determines the specificity of
 +
the transcription factor which enables the dCas9-VP64 to activate different genes
 +
depending on the sequence of the gRNA molecule and the promoter. gRNA consists of two
 +
parts, a scaffold and a 20 bp Specificity Determinant Sequence (SDS) on the 5' end.
 +
The scaffold constitutes the majority of the gRNA molecule and gives it its structure
 +
whereas the SDS binds to the target site in the gene promoter.  Cyclins are proteins
 +
that are involved in the progression of the cell cycle, therefore activated at specific
 +
times [5]. To mimic the specific production pattern of cyclins the dCas9-VP64 gene is
 +
placed under the control of a yeast cyclin promoter. Therefore the Cas9 will be
 +
produced in the G1 phase. When the Cas9 and gRNA dimerize and the transcription factor
 +
is activated, it in turn activates the transcription of a new fluorescent protein and
 +
a new gRNA molecule to act as a memory for the next cycle. Once this age counter is
 +
implemented, it would be possible to sort cells according to their replicative age
 +
automatically with a flow cytometer device.
 +
</p>
 +
<h4>References</h4>
 +
<ol id="references">
 +
<li>Hackett, E.A., et al., A family of destabilized cyan fluorescent proteins as  
 +
transcriptional reporters in S. cerevisiae. Yeast, 2006. 23(5): p. 333-349.</li>
 +
 +
<li>Andersen, J.B., et al., New unstable variants of green fluorescent protein for
 +
studies of transient gene expression in bacteria. Applied and environmental microbiology,
 +
1998. 64(6): p. 2240-2246.</li>
 +
 +
<li>Moon, T.S., et al., Genetic programs constructed from layered logic gates in single
 +
cells. Nature, 2012. 491(7423): p. 249-253.</li>
-
<li>Andersen, J.B., et al., New unstable variants of green fluorescent protein for
+
<li>Farzadfard, F., S.D. Perli, and T.K. Lu, Tunable and multifunctional eukaryotic
-
studies of transient gene expression in bacteria. Applied and environmental microbiology,  
+
transcription factors based on CRISPR/Cas. ACS synthetic biology, 2013. 2(10): p.  
-
1998. 64(6): p. 2240-2246.</li>
+
604-613.</li>
-
<li>Moon, T.S., et al., Genetic programs constructed from layered logic gates in single
+
<li>Nasmyth, K., At the heart of the budding yeast cell cycle. Trends in Genetics,
-
cells. Nature, 2012. 491(7423): p. 249-253.</li>
+
1996. 12(10): p. 405-412</li>
 +
</ol>
-
<li>Farzadfard, F., S.D. Perli, and T.K. Lu, Tunable and multifunctional eukaryotic
 
-
transcription factors based on CRISPR/Cas. ACS synthetic biology, 2013. 2(10): p.
 
-
604-613.</li>
 
-
 
-
<li>Nasmyth, K., At the heart of the budding yeast cell cycle. Trends in Genetics,
 
-
1996. 12(10): p. 405-412</li>
 
-
</ol>
 
-
</td>
 
-
</tr>
 
-
 
-
<tr>
 
-
<td width="45%"  valign="top">
 
-
<p><b>Super Mega Ultra Awesome!!!</b> (....and classified until now.)<br>(1-2 Paragraphs will be seen here soon)</p>
 
-
<br>
 
-
<h3>References </h3>
 
-
<p>
 
-
iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you. </p>
 
-
</td>
 
-
 
-
<td></td>
 
-
<td  width="45%"  valign="top">
 
<p> You can use these subtopics to further explain your project</p>
<p> You can use these subtopics to further explain your project</p>
-
 
<ol>
<ol>
<li>Overall project summary</li>
<li>Overall project summary</li>
Line 126: Line 118:
<li>Conclusions</li>
<li>Conclusions</li>
</ol>
</ol>
-
 
<p>
<p>
-
It's important for teams to describe all the creativity that goes into an iGEM project, along with all the great ideas your team will come up with over the course of your work.  
+
It's important for teams to describe all the creativity that goes into an iGEM project, along with
 +
all the great ideas your team will come up with over the course of your work.  
</p>
</p>
-
 
<p>
<p>
-
It's also important to clearly describe your achievements so that judges will know what you tried to do and where you succeeded. Please write your project page such that what you achieved is easy to distinguish from what you attempted.  
+
It's also important to clearly describe your achievements so that judges will know what you tried
 +
to do and where you succeeded. Please write your project page such that what you achieved is easy  
 +
to distinguish from what you attempted.  
</p>
</p>
-
</td>
 
-
 
-
</tr>
 
-
 
-
 
-
</table>
 
</div>
</div>
</body>
</body>

Revision as of 14:05, 13 August 2014

TemplateUp

Project Description

Nowadays the determination of replicative age of yeast cells is done by counting the budding scars of each cell in a microscope, a process time consuming and not effective. Our team goal with the iGEM project is to construct a yeast generation counter. The idea is that each time the cell divides a different florescent protein [1, 2] is produced. Therefore by examining the cell under a microscope or in a flow cytometer one can determine how many times the cell has divided. This would be achieved by constructing a logical AND gate in the cell [3] where the input signals consist of a cyclin activated dCas9-VP64 and a guide RNA (gRNA) [4] signal from the previous cell cycle. The output response consist of a different fluorescent protein and a new gRNA molecule, see figure 1.


Figure 1. Schematic representation of the logical AND gate with the input and output signals.

dCas9-VP64 is an engineered turntable transcription factor only active when dimerized with an interchangeable gRNA molecule. The gRNA also determines the specificity of the transcription factor which enables the dCas9-VP64 to activate different genes depending on the sequence of the gRNA molecule and the promoter. gRNA consists of two parts, a scaffold and a 20 bp Specificity Determinant Sequence (SDS) on the 5' end. The scaffold constitutes the majority of the gRNA molecule and gives it its structure whereas the SDS binds to the target site in the gene promoter. Cyclins are proteins that are involved in the progression of the cell cycle, therefore activated at specific times [5]. To mimic the specific production pattern of cyclins the dCas9-VP64 gene is placed under the control of a yeast cyclin promoter. Therefore the Cas9 will be produced in the G1 phase. When the Cas9 and gRNA dimerize and the transcription factor is activated, it in turn activates the transcription of a new fluorescent protein and a new gRNA molecule to act as a memory for the next cycle. Once this age counter is implemented, it would be possible to sort cells according to their replicative age automatically with a flow cytometer device.

References

  1. Hackett, E.A., et al., A family of destabilized cyan fluorescent proteins as transcriptional reporters in S. cerevisiae. Yeast, 2006. 23(5): p. 333-349.
  2. Andersen, J.B., et al., New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Applied and environmental microbiology, 1998. 64(6): p. 2240-2246.
  3. Moon, T.S., et al., Genetic programs constructed from layered logic gates in single cells. Nature, 2012. 491(7423): p. 249-253.
  4. Farzadfard, F., S.D. Perli, and T.K. Lu, Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS synthetic biology, 2013. 2(10): p. 604-613.
  5. Nasmyth, K., At the heart of the budding yeast cell cycle. Trends in Genetics, 1996. 12(10): p. 405-412

You can use these subtopics to further explain your project

  1. Overall project summary
  2. Project Details
  3. Materials and Methods
  4. The Experiments
  5. Results
  6. Data analysis
  7. Conclusions

It's important for teams to describe all the creativity that goes into an iGEM project, along with all the great ideas your team will come up with over the course of your work.

It's also important to clearly describe your achievements so that judges will know what you tried to do and where you succeeded. Please write your project page such that what you achieved is easy to distinguish from what you attempted.

Retrieved from "http://2014.igem.org/Team:Gothenburg/Project"