Team:Gothenburg-Sweden/Project

From 2010.igem.org

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<b>• Working package 14: Apply for funding</b><br>
<b>• Working package 14: Apply for funding</b><br>
Also running alongside the other WPs. Funding is initially applied at science funding committees and later, as there are results to be presented, at major companies interested in the possibilities presented by this project. Funding should cover laboratory expenses, travel to Boston and team t-shirts.<br><br></td></tr>
Also running alongside the other WPs. Funding is initially applied at science funding committees and later, as there are results to be presented, at major companies interested in the possibilities presented by this project. Funding should cover laboratory expenses, travel to Boston and team t-shirts.<br><br></td></tr>
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<td class="text5">Theoretical background</td>
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<td><p>Cellular stress is sensed  by a key protein called AMP-activated protein kinase (AMPK). The AMPK protein  complex is conserved among all eukaryotes, including yeast, plants and humans.  In humans this is the target of most anti-diabetic drugs in the market today  and is also implicated in many other metabolic disorders such as obesity and  atherosclerosis and also in developmental processes such as cell cycle and  ageing, etc. In yeast, this protein is called SNF1.</p>
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<p>This protein functions as a heterotrimer  consisting of α, β and γ subunits. The α-subunit contains the catalytic domain  (actual kinase) while the β-subunit is the regulatory domain. It response to  high levels of AMP and undergoes a conformational change, which exposes the  phosphorylation site on the catalytic α-subunit. AMPK is activated by  phosphorylation on this conserved site on the α-subunit (shown in the figure below).<br>
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                        <img width="590" height="477" src="https://static.igem.org/mediawiki/2010/b/b9/Description-pic.JPG" align="left" hspace="12" alt="IGEM project description2.bmp"><br>
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                      </p>
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                    <p>&nbsp;</p>
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                    <p class="STYLE3">Figure 1: (1)Schematic representation of the  AMPK heterotrimer. The figure on the left shows the conformation in an inactive  state. On the right, the conformational change in the γ-subunit triggers a  structural change in the α-subunit, which is phosphorylated. (2) Schematic  representation (stereo view) of the heterotrimer core of SNF1. The regulatory sequence  of the a-subunit  (Snf1) is shown in red and  the rest is in yellow; theGBDof the b-subunit (Sip2) is shown in cyan and the  rest is in magenta; and the c-subunit (Snf4) is shown in green. The positions  of AMP (stick model in black), as observed from our studies and in the S. pombe  enzyme9, as well as that of b-cyclodextrin (in grey) as bound in the rat GBD10,  are shown for reference.</p>
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<td class="text5">techniques</td>
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  <td><p class="STYLE5">Fusion PCR:</p>
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    <p>Fusion PCR is a method used to ligate strands  of DNA without using restriction enzymes or ligase (Horton 1989). For a three  fragment fusion six primers is necessary: Two ordinary primers and four fusion  primers. The fusion primers should optimally have at least 20 bp homology with  the next fragment (figure.2) and fusion efficiency is highly dependent of  GC-content within these regions (Kamochai et al. 2008). In other aspects than  the fusion primer design and the number of steps to produce a final product  fusion PCR follow ordinary PCR protocol.</p>
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    <p><img src="https://static.igem.org/mediawiki/2010/c/cb/Project_clip_image002.jpg" alt="Fusion PCR" width="504" height="392"><br>
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      <span class="STYLE3">Figure 2: Essential steps for fusing three  DNA fragments using fusion PCR<br>
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      1-3: Each fragment is run with primers that results in regions of homology  corresponding to the next piece.<br>
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      4: Two fragments with homology are run together, resulting in fusion and  amplification. <br>
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      5: The product from step 4 is run with the last homology fragment, resulting in  fusion and amplification of the final product. </span><br>
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    <p>References:</p>
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    <ul>
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      <li>Kamonchai,  Cha-aim., Tomoaki, Fukunaga., Hisashi, Hoshida &amp; Rinji Akada (2009)  Reliable fusion PCR mediated by GC-rich overlap sequences. <em>Gene,</em> 434: 43-49.</li>
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          <li>Horton, Robert., Hunt, Henry., Ho, Stefan., Pullen, Jeffrey &amp; Pease,  Larry<strong> </strong>(1989)  Engineering<strong> </strong>hybrid genes without the use of restriction enzymes:<strong> </strong>gene  splicing by overlap extension.<strong> </strong><em>Gene</em>,  77:61-68.<strong></strong></li>
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    <p>&nbsp;</p>
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    <h3 class="STYLE5">FRET:</h3>
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    <p>&nbsp;</p>
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    <p>FRET  (Förster resonance energy transfer) is an electrodynamic phenomenon that occurs  between two chromophores, where one is a donor and the other one is an  acceptor. The donor molecule, in its excited state, transfers energy to the  acceptor molecule through non-radiative dipole-dipole interactions and does not  involve the appearance of a photon. The rate of energy transfer between the  donor and acceptor depends on the spectral overlap, the quantum yield of the  donor, the relative orientation of the transition dipoles and the distance  between the molecules. Owing to the distance dependence, it is possible to  measure distances between donor molecules and acceptor molecules. The most  common application of FRET is to measure the distance between two sites on a  macromolecule, typically a protein is covalently labeled with two chromophores.  In the case of multi-domain protein, FRET can be used to measure conformational  changes that move the domains closer or further apart. <br>
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        The rate of  energy transfer from a donor to an acceptor, kT(r) is given by the  expression:</p>
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                                  <img src="https://static.igem.org/mediawiki/2010/a/a8/Project_clip_image002.gif" alt="FRET1" width="102" height="48">          eq 1 <br>
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    <p>In equation  1, τD is the lifetime of the donor in the absence of acceptor and r  is the donor to acceptor distance. The distance at which the resonance energy  transfer efficiency is 50 % is called the Förster distance, R0. At  this distance, half of the donor molecules are decaying by energy transfer and  half are decaying by the radiative and non-radiative rates. This distance is  typically in the rage of 20 to 60 Å. The energy transfer efficiency (E) is the  fraction of energy absorbed by the donor which is transferred to the acceptor.  This fraction is given by equation 2.</p>
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                                  <img src="https://static.igem.org/mediawiki/2010/1/1a/Project_clip_image004.gif" alt="FRET2" width="81" height="48">                 eq 2 <br>
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    <p>Combining  these two equations gives an expression for how the energy transfer efficiency  varies as the inverse sixth power of the distance between the donor and  acceptor molecules, equation 3 (Lakowicz,  2006).</p>
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                                  <img src="https://static.igem.org/mediawiki/2010/d/d5/Project_clip_image006.gif" alt="FRET3" width="76" height="48">                eq 3 <br>
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    <p>The expression is graphically demonstrated  in figure 3. Because of the  inverse sixth power dependence on the distance between the donor and acceptor  molecule, the curve has a very sharp decline. When designing a biochemical experiment the  distance between the donor and acceptor molecule is of great importance. The useful range for measuring FRET is between 0.5 times R0  and 1.5 times R0. For most applications in cell biology, FRET  experiments have a relatively binary readout. Measurements will often only be  able to distinguish between high-FRET  and low-FRET, or simply between  the presence and absence of FRET (Kremers, Piston and Davidson,  n.d.).</p>
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    <p>&nbsp;</p>
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    <p><img src="https://static.igem.org/mediawiki/2010/0/0b/Project_clip_image008.jpg" alt="FRET4" width="375" height="281"> <br>
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                                    <span class="STYLE3">Figure 3:  Dependence of energy transfer efficiency (E) on donor to acceptor  distance (r).</span></p>
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    <p>&nbsp;</p>
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    <p>References:</p>
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      <li>Kremers,  G.-J., Piston, D.W. and Davidson, M.W. <em>Microscopyu</em>, [Online], Available: <a href="http://www.microscopyu.com/articles/fluorescence/fret/fretintro.html">http://www.microscopyu.com/articles/fluorescence/fret/fretintro.html</a> [09 Jul 2010].<br>
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        </li>
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          <li>Lakowicz, J.R. (2006) <em>Principles of Fluorescence  Spectroscopy</em>, Third edition edition, New York: Springer.</li>
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<td class="text5">snf1 &amp; ampk </td>
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  <td><h1 class="STYLE5">SNF1:</h1>
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    <p>&nbsp;</p>
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    <p>SNF1  belongs to a family of protein kinases which is highly conserved in eukaryotes  (Hedbacker&nbsp;2009). The SNF1 homologue in mammalian cells is called AMP-  activated protein kinase (AMPK) and it regulates energy balance in mammals. It  is therefore an attractive target for drug discovery against for example  diabetes and obesity (Amodeo 2007). SNF1 was identified in <em>Saccaromyces cerevisiae </em>in 1981 when the snf1 mutation was found  (Carlson 1981). <br>
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      The main  function of SNF1 is to help yeast cells adapt to glucose limitation and it is  required for growth on other carbon sources than glucose such as sucrose  (Hedbacker 2009). This is how SNF got its name, since SNF is an abbreviation  for sucrose-nonfermenting (Hong 2007). SNF1 is consequently important in response  to metabolic stress and it also regulates protective mechanisms against  different stresses in response to glucose depletion (Hong 2007). Besides the  primary role in nutrient stress, it is also important in environmental stresses  including sodium ion stress, heat shock, alkaline pH, oxidative stress and  genotoxic stress (Hedbacker 2009).  SNF1  regulates transcription of many genes and controls the activity of metabolic  enzymes involved in fatty acid metabolism and carbohydrate storage (Hong 2007).  SNF1 also controls nutrient-responsive cellular developmental processes such as  meiosis, sporulation and aging (Hedbacker&nbsp;2009).<br>
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      SNF1 is  activated when phosphorylated by the upstream kinases Sak 1, Tos 3 and Elm 1  (Hedbacker&nbsp;2009). Each of all three kinases is enough to activate SNF1 in  response to stress but Sak1 has the major role (Hong&nbsp;2007). SNF1 is  activated by glucose limitation but the glucose signal regulating activation of  SNF1 is not known (Hong 2007). Hong <em>et  al.</em> studied mutants lacking all three kinases and their result was that  SNF1 still was activated in response to sodium ion and alkaline stress which  according to Hong <em>et al.</em> suggests  that stress signals might activate SNF1 by a mechanism that is independent of  the upstream kinases. SNF1 is inactivated by the Ref1-GLc7 protein phosphatase  1 (Hedbacker 2009). The SNF1 protein kinase pathway is robust and low levels of  SNF1 activity is mostly enough for growth under many different stress  conditions (Hong 2007). Apart from activation and deactivation, regulation of  SNF1 is also achieved through autoinhibition and through control of the  subcellular localization (Hedbacker&nbsp;2009). </p>
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    <h2 class="STYLE5">Subunit structure and  function: </h2>
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    <p>Like its  ortholog AMPK, SNF1 is heterotrimeric, which means that it consists of three  subunits. The yeast genome encodes the catalytic alpha subunit Snf1, three  different beta subunits, Sip1, Sip2 and Gal83, and the gamma subunit Snf4  (Hedbacker&nbsp;2009). The heterotrimeric interface is located at residue  531-586 on Snf1, residues 375-412 of Sip2 and residues 38-45 of Snf4 (Amodeo, Rudolph, &amp; Tong, 2007).</p>
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    <h3 class="text3">Snf1- alpha subunit:</h3>
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    <p>&nbsp;</p>
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    <p>This  catalytic subunit spans 633 amino acids and contains 3 major features, namely  the kinase domain, the activation site and a regulatory domain (Hedbacker  2009). The regulatory domain consists of the AIS-domain (Auto Inhibitory  Sequence) which functions by binding to the kinase domain and thereby silencing  the catalytic activity. The kinase domain also contains the activation site of  the full protein complex, Thr210, which is phosphorylated by upstream kinases  when the cells are exposed to external stress (McCartney &amp;  Schmidt 2001).</p>
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    <h3 class="text3">Beta- subunits:</h3>
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    <p>There are  three known types of the beta subunit, Sip1, Sip2 and Gal83 (Hedbacker 2009).  All three beta subunits in yeast share a conserved GBD (glucogen binding  domain) with their common mammalian ortholog AMPK (Wiatrowski et al 2004). The  three different subunits differ in their cellular localization when glucose is  limited. Sip1 localizes to the vacuolar membrane, Sip2 remains cytoplasmic  while Gal83 localizes to the nucleus (Vincent <em>et al </em>2001). Due to this the subunits contain a few differing  elements, for example the Gal83-subunit contains a nuclear localization signal  on the c-terminus (Hedbacker and Carlson 2006)</p>
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    <p>.</p>
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    <h3 class="text3">Snf4 – gamma subunit:</h3>
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    <p>&nbsp;</p>
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    <p>The  Snf4-subunit consits of 2 bateman domains made up of CBS (cystathionine-beta  synthase) repeats that enable them to bind to adenosine derivatives (Hedbacker  2009).  This subunit is also a part of  the SNF1 activity regulating machinery through binding to the AIS -region of  the Snf1 subunit when the glucose supply is low. This binding inhibits the  autoinhibition of the kinase activity performed by the AIS sequence (Celenza  and Carlson 1989). <br>
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      <li>       Carlson,  M., Osmond, B. C., Botstein, D. (1981) Mutants of yeast defective in sucrose  utilization. <em>Genetics, </em>98:25-40.<br>
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          <li>Celenza, J. L.,  Carson, M. (1989) Mutational Analysis of the Saccharomyces cerevisiae SNF1  Protein<br>
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          Kinase and Evidence for  Functional Interaction with the SNF4 Protein. <em>Molecular and cellular biology </em>9: 5034-5044</li>
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      <li>Hedbacker,  K., Carlson, M. (2009) SNF1/AMPK pathways in yeast. <em>Frontiers in Bioscience, </em>13:2408‑2420.  <br>
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          </li>
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      <li>Hedbacker,  K., Carlson, M. (2006) Regulation of the Nucleocytoplasmic Distribution of  Snf1-Gal83 Protein Kinase. Eukaryotic Cell<em>, </em>5: 1950-1956.  <br>
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          </li>
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      <li>Amodeo, G.  A., Rudolph, M. J., Tong, L. (2007) Crystal structure of the heterotrimer core  of Saccaromyces cerevisiae AMPK homologue SNF1. <em>Nature</em>, 449:492-495.<br>
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          </li>
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      <li>Hong, S-P.,  Carlson, M. (2007) Regulation of Snf1 Protein Kinase in Response to  Environmental Stress. <em>The Journal of  Biological Chemistry, </em>282:16838-16845.<br>
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      <li>McCartney,  R. R , Schmidt, C. S. (2001) Regulation of Snf1 Kinase Activation requires  phosphorylation of threonine 210 by an upstream kinase as well as a distinct  step mediated by the Snf4 subunit<strong>. </strong><em>The Journal of Biological Chemistry, </em>276:36460-36466<br>
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                                    </li>
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      <li><strong>Vincent,  O., Townley, R., Kuchin, S. and Carlson, M. (2001)</strong> Subcellular localization of the  Snf1 kinase is regulated by specific&nbsp;β subunits and a novel glucose  signaling mechanism.  <em>Genes and Development </em>15; 1104-1114<br>
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                                    </li>
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      <li><strong>Wiatrowski,  H. A., van Denderen, B. J. W, Berkey, C. D., Kemp, B. E., Stapleton, D. and  Carlson, M. (2004) </strong>Mutations in the Gal83 Glycogen-Binding Domain Activate the Snf1/Gal83  Kinase Pathway by a Glycogen-Independent Mechanism. <em>Molecular and Cellular Biology</em> 24; 352-361</li>
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<td class="text5">safety questions </td>
<td class="text5">safety questions </td>

Revision as of 11:05, 14 July 2010

Chalmers University of Technology

 
The project
Synthetic readout of cellular stress
 
 
Project Description
 

The conceptual idea is to use the conformational change in the SNF1 protein complex to establish a FRET (Förster Resonance Energy Transfer) system for cellular stress detection. There are two chromophores tagged at appropriate locations and upon undergoing the conformational change, they come in close proximity such that the emission energy from one can excite the other, thus resulting in different emission energy. There are two possibilities to be tested:

1. To fuse the chromophores to the subunits of the Snf1 complex to build an active readout of the conformational change
2. To fuse the chromophores to a short peptide (SAMS peptide) that can be phosphorylated by Snf1 and is commonly used as a read-out of Snf1 activity.


• Working package 1: Find positions for fluorescent markers in SNF1
For WP1 all present information about the protein complex is utilized, so that the fluorescent tags do not interfere with active sites or evolutionary conserved regions. Two alternatives should be investigated and presented with regard to present information and distance between fluorophores. The working package is ended with a 3D modeling of the tagging suggestions to further investigate functionality.

• Working Package 2: Decide what fluorescent protein to use
To apply FRET a compatible fluorphore pair is necessary. Also, efficiency and duration in respect to e.g. photonic bleaching should be taken into consideration.

• Working package 3: Find DNA sequence for SAMS-peptide
Beside the two tagging alternative from WP1 a SAMS-peptide will be used. Both ends will be tagged with fluorophores and conformational change should be indicated with FRET-signal during conformational change at phophorylation by active SNF1.

• Working package 4: Decide on plasmid backbone
The plasmid containing fusion protein should be able to amplify in E.coli and also contain a strong promoter for protein expression in yeast. Use VectorNTI to find good alternatives and locate RE sites.

• Working package 5: Design primers for fusion PCR
When WP 1-4 is finished the primer design can be initiated. Primers should be designed so that they have a high melting temperature, preferably around 60°C, with at least 20 bp homology with each side that should be fused. Primers at utter end of the fusion proteins should include restriction enzyme sites with GGCC repetition at the end. After rigorous proofreading the primers should be ordered together with a primer representing the SAMS peptide.

• Working package 6: Acquire plasmid backbone for transfection with fusion protein
Look up how the plasmid backbone from WP4 can be acquired. If it is a common backbone it should be avaible in the plasmid bank at the laboratory, otherwise it should be ordered.

• Working package 7: Perform fusion PCR
When the primers from WP5 has arrived the fusion PCR can be initiated. As a template for the protein subunits in SNF1 genomic DNA from yeast should be used, this should be avaible at the laboratory, and as template for fluorescent proteins dried DNA provided by iGEM will be used. Create a PCR mastermix that includes all ingredients except the DNA template. Each fusion is run in five steps according to the manual and each step is analyzed on agar gel so that the correct fragments are produced.

• Working package 8: Ligate insert into plasmids
After WP7 is successful the fusion protein should be inserted into the plasmid backbone. Use restriction enzymes as decided in WP6 and follow ligation protocol. Afterwards the plasmids should be purified with miniprep, they should be cut again and put on a gel to verify that the insert was successful.

• Working package 9: Amplify transfection plasmids
After WP8 the plasmid need to be amplified before being tranfected into yeast. Tranfect E.coli and amplify over night. Purify the amplified plasmids with a miniprep kit.

• Working package 10: Sporulate diploid yeast strains to acquire Snf1 deletion mutants
Grow diploid yeast strains with Snf1 deletions on sporulation plats to acquire haploid deletion mutants. Use protocol to clear the population from diploids and unwanted haploids.

• Working package 11: Transfect yeast with tagged fusion protein
After WP9-10 the hapoloid yeast deletion strain should be tranfected with the fusion protein plasmid. When the yeast has recovered from transfection the strain should be grown on sucrose overnight to show transfection result.

• Working package 12: Apply FRET and analyze results
By using a microscopy with FRET set up the success of previous WP can be concluded from the presence or absence of a FRET signal. Analyze results and make conclusions together with Marcus Wilhelmsson.

• Working package 13:Write report
WP 13 is running alongside with the other WPs during the whole project. Initially aims and theory is the major work load and as the project progress results and analysis claims the major part of this WP.

• Working package 14: Apply for funding
Also running alongside the other WPs. Funding is initially applied at science funding committees and later, as there are results to be presented, at major companies interested in the possibilities presented by this project. Funding should cover laboratory expenses, travel to Boston and team t-shirts.

safety questions
 
 

to be continued

 

 

 
 
 
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