Difference between revisions of "Team:Linkoping Sweden/Description"

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            <h3>Our Workplan</h3>
 
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            <!--<h3>Project design</h3>-->
 
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We designed a superduper-plasmid with the code for three different chaperones, GroES/GroEL, DnaK and Trigger factor each with a different promotor. The purpose of this was to be able to induce transcription of the specific chaperones in different combinations. Four other plasmids were designed for the substrates; E-GFP bound to Amyloid-beta or Tau and M-Neongreen protein bound to Amyloid-beta or Tau, for these substrates we used a fourth promotor. After some research, we found that both E-GFP and M-Neongreen are suitable fusion proteins for proteins that are hard to express in E.coli and decided to use both.</p> 
 
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<p>The plasmid with chaperones and the plasmid with substrates have different antibiotic resistance genes to make it possible to select the bacteria’s that expresses both plasmids. </p>
 
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<p>We will induce the expression of the chaperones before we induce the expression of the substrates to make sure that there are chaperones present when the translation of the substrates begins. </p>
 
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Both fusion proteins are fluorescent which will enable us to get an indication of the expression. Further we will use several methods to detect and quantify our proteins.  </p>
 
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                The overview of the project is summarized in figure 1 below. As can be seen in the figure, chaperones and conditions will be tested with various experiments, with the purpose to find the best setup for further detailed studies. The detailed studies will involve modeling with systems biology, where the modeling team will give suggestions on what the laboratory team shall do to achieve the desired task. The flow of data between the laboratory and the modeling team will eventually result in the final data, with is the optimized setup for the expression of our peptides.</p>
 
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            <img class="content_image" src="https://static.igem.org/mediawiki/2017/5/5c/T--Linkoping_Sweden--Updated_detailed_flow-chart.png" alt="Figure 1. Detaield flow chart of the project."/>
 
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            <center><i>Figure 1. Detailed design of the project.</i></center>
 
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            <!-- <h1>Experiments</h1> -->
 
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            <!--År 2016 var det skrivet vad som gjordes under respektive labbdag.-->
 
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          <!--  <h1>Protocols</h1> -->
 
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            <!--Verkar enligt LiU iGEM 2016 vara vilka metoder och lösnignar mm. man använt.-->
 
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            <!-- <h1>Results</h1> -->
 
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            <!--Slutgiltiga reultat av labbandet.-->
 
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            <!-- <h1>Safety</h1> -->
 
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            <!--Relevant att ha med, t ex säkerhet kring organismer och kemikalier, se LiU iGEM 2016-->
 
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            <!-- <h1>Notebook</h1> -->
 
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            <!--Tidslinje av projektet, från starten i mars till Boston -->
 
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            <!-- <h1>Economic viability</h1> -->
 
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            <!--Mer anpassat till å 2016-->
 
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<h1> Project description</h1>
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The aim of the project is to optimize the expression of the proteins amyloid-beta and tau in the bacteria Escherichia coli. These proteins are known to be linked to the development of Alzheimer's disease, though the cause of the disease is still unknown [1]. They are both found in the brain in and around our neurons, and during Alzheimer's disease they accumulate to form plaques and tangles [3, 4]. Because these proteins are prone to misfolding and aggregate they are hard to study in a laboratory environment, which has slowed down research. It’s here our project comes in, trying to facilitate the production of these proteins.
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To optimize the expression of the proteins we will be using chaperones. Chaperones are proteins that can help other proteins to fold into their native three-dimensional shape, by dissolving aggregates and guiding both unfolded and misfolded proteins to their correctly folded form. During our project we will be using four different chaperones and we will see how different combinations of these affect the expression of Amyloid-beta and Tau respectively.
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The chaperones we will study are GroEL, GroES, DnaK and Trigger factor. GroEL and GroES helps unfolded and misfolded proteins to fold correctly. DnaK helps with disaggregating and with unfolding of misfolded proteins. And Trigger factor helps the protein to remain unfolded during its synthesis.
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</br>To express the chaperones in the bacteria we will use a large plasmid containing all four chaperones and give them unique promotors, one for GroEL-GroES, one for DnaK and one for TF. The purpose of this was to easily be able to induce transcription of the specific chaperones in different combinations.
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<a class="content-button" href="#"> Read more about the different chaperones here </a>
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To measure our expression of these proteins we will fuse them with fluorescent proteins called E-GFP and mNeonGreen. Thereby we can measure the fluorescence to follow the expression of the fusion proteins. For every one of alzheimer - fluorescent fusion protein we will create a plasmid resulting in 4 combinations. These 4 plasmids will use a fourth promotor so that we can control this expression as well.
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<a class="content-button" href="#"> Read more about the fusion proteins here </a>
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The overview of the project is summarized in figure 1 below. As can be seen in the figure, chaperones and conditions will be tested in a screening process, with the purpose to find the best setup for further detailed studies. The detailed studies will involve modeling with systems biology, where the modeling team will give suggestions on what the laboratory team shall do to achieve the desired task. The flow of data between the laboratory and the modeling team will eventually result in the final data, with is the optimized setup for the expression of our peptides.
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<p> <i> Figure 1. An overviewing design of the project. </i> </p>
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1. Alzheimerfonden. Alzheimers sjukdom [Internet]. Cited 2017-06-15. Available from: http://www.alzheimerfonden.se/om_demens/alzheimers_sjukdom [in swedish]
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</br> 2. Alzheimer´s Disease International. Dementia statistics [Internet]. Cited 2017-06-27. Available from: https://www.alz.co.uk/research/statistics
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</br> 3. Alzheimerfonden. Lexikon för Alzheimers sjukdom och andra demenssjukdomar [Internet]. Cited 2017-06-15. Available from: http://www.alzheimerfonden.se/om_demens/lexikon [in swedish]
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</br> 4. Bloom GS. Amyloid-β and TauThe Trigger and Bullet in Alzheimer Disease Pathogenesis. JAMA Neurol. 2014;71(4):505-508. doi:10.1001/jamaneurol.2013.5847
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Revision as of 08:07, 3 August 2017

Project description

The aim of the project is to optimize the expression of the proteins amyloid-beta and tau in the bacteria Escherichia coli. These proteins are known to be linked to the development of Alzheimer's disease, though the cause of the disease is still unknown [1]. They are both found in the brain in and around our neurons, and during Alzheimer's disease they accumulate to form plaques and tangles [3, 4]. Because these proteins are prone to misfolding and aggregate they are hard to study in a laboratory environment, which has slowed down research. It’s here our project comes in, trying to facilitate the production of these proteins.

To optimize the expression of the proteins we will be using chaperones. Chaperones are proteins that can help other proteins to fold into their native three-dimensional shape, by dissolving aggregates and guiding both unfolded and misfolded proteins to their correctly folded form. During our project we will be using four different chaperones and we will see how different combinations of these affect the expression of Amyloid-beta and Tau respectively.

The chaperones we will study are GroEL, GroES, DnaK and Trigger factor. GroEL and GroES helps unfolded and misfolded proteins to fold correctly. DnaK helps with disaggregating and with unfolding of misfolded proteins. And Trigger factor helps the protein to remain unfolded during its synthesis.
To express the chaperones in the bacteria we will use a large plasmid containing all four chaperones and give them unique promotors, one for GroEL-GroES, one for DnaK and one for TF. The purpose of this was to easily be able to induce transcription of the specific chaperones in different combinations.

To measure our expression of these proteins we will fuse them with fluorescent proteins called E-GFP and mNeonGreen. Thereby we can measure the fluorescence to follow the expression of the fusion proteins. For every one of alzheimer - fluorescent fusion protein we will create a plasmid resulting in 4 combinations. These 4 plasmids will use a fourth promotor so that we can control this expression as well.

The overview of the project is summarized in figure 1 below. As can be seen in the figure, chaperones and conditions will be tested in a screening process, with the purpose to find the best setup for further detailed studies. The detailed studies will involve modeling with systems biology, where the modeling team will give suggestions on what the laboratory team shall do to achieve the desired task. The flow of data between the laboratory and the modeling team will eventually result in the final data, with is the optimized setup for the expression of our peptides.

project flow-chart

Figure 1. An overviewing design of the project.


1. Alzheimerfonden. Alzheimers sjukdom [Internet]. Cited 2017-06-15. Available from: http://www.alzheimerfonden.se/om_demens/alzheimers_sjukdom [in swedish]
2. Alzheimer´s Disease International. Dementia statistics [Internet]. Cited 2017-06-27. Available from: https://www.alz.co.uk/research/statistics
3. Alzheimerfonden. Lexikon för Alzheimers sjukdom och andra demenssjukdomar [Internet]. Cited 2017-06-15. Available from: http://www.alzheimerfonden.se/om_demens/lexikon [in swedish]
4. Bloom GS. Amyloid-β and TauThe Trigger and Bullet in Alzheimer Disease Pathogenesis. JAMA Neurol. 2014;71(4):505-508. doi:10.1001/jamaneurol.2013.5847