Team:TU-Eindhoven/LEC/LabTheory

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<h3>Design challenge</h3>
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[[File:Plasmids.jpg|710px]]
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<p>The aim of this project is to design and produce a new multi-colored display in which genetically engineered cells function as pixels analogous to a conventional display. In the lab we will create cells that emit light in response to an electric stimulus. This can be achieved by <span class= "lightblue">genetic modification of yeast cells</span>, through the introduction of fluorescent calcium sensors and utilizing the already present calcium channels. Already anticipating on the probably slow response time of our 'pixel' we also intend to over express the calcium channels together with the expression of the GECO proteins as a means to enhance the calcium flux. On top of our great endeavor an other innovative aspect is the fact that our system responds to <span class= "lightblue">exogeneous stimuli</span> instead of the more traditional systems which respond to intracellular processes.</p>
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<p>Fig. 1 Yeast cell with the needed plasmids</p>
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<h3>Design challenge</h3>
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<p>The plasma membrane of the brewer's yeast <i>Saccharomyces cerevisiae</i> contains the <span class= "lightblue">CCH1-MID1 channel</span> that is homologous to mammalian voltage-gated calcium channels (VGCCs). It is hypothesized that upon depolarization of the plasma membrane <span class= "lightblue">calcium ions selectively enter the cytoplasm</span> through these channels<html><a href="#ref_Iida"name="text_Iida"><sup>[1]</sup></a></html>. Light will be emitted by fluorescence of the <span class= "lightblue">GECO protein</span><html><a href="#ref_zhao"name="text_zhao"><sup>[2]</sup></a></html>, a calcium dependent fluorescent protein that is expressed from a genetically engineered plasmid. When this calcium sensor is exposed to an elevated intracellular calcium concentration its fluorescence will increase significantly; consequently when the calcium concentration drops its fluorescence will diminish. The calcium can enter the cell's cytoplasm upon electrical stimulation of the calcium channel, after which the GECO protein will start to fluoresce. Finally the excess of calcium will be removed by active transport within the cell to restore it's <span class= "lightblue">homeostatic level</span>, and the amount of fluorescence will decrease.</p>
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<p>The aim of this project is to design and produce a new multi-color display in which genetically engineered cells function as pixels analogous to how a flat panel display works. In the lab we will make living cells that emit light in response to an electric stimulus. This can be achieved by genetic modification of yeast cells, through the introduction of fluorescent calcium sensors and calcium channels. The plasma membrane of the brewer's yeast <i>Saccharomyces cerevisiae</i> contains the CCH1-MID1 channel that is homologous to mammalian voltage-gated calcium channels (VGCCs). It is hypothesized that upon depolarization of the plasma membrane, calcium ions selectively enter the cytoplasm through these channels <html><aref="#ref_Iida"name="text_Iida"><sup>[1]</sup></a></html>. Light will be emitted through the fluorescence of the GECO protein <html><ahref="#ref_zhao"name="text_zhao"><sup>[2]</sup></a></html>, a calcium sensor that is expressed from a genetically engineered plasmid. When the calcium concentration is high, the GECO protein will fluoresce, but when it is very low, the protein will not fluoresce. Electrical stimulation of the cell will allow calcium to enter into the cytoplasm through the calcium channels and the GECO proteins will start to fluoresce. After a while the calcium concentration will drop to homeostatic levels through active transport of calcium ions by the yeast's vacuole and the fluorescence will cease. Challenges in the laboratory can be found in creating yeast cells with both GECO proteins and a sufficient number of calcium channels incorporated.</p>
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<p>Challenges in the laboratory can be found in creating yeast cells with the GECO protein as well as a sufficient amount of calcium channels which consist of two separate proteins. The important design choices regarding the biological work are further motivated in the text below.</p>
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<h3>Design choices</h3>
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<div class="vectorImage">[[File:Plasmids.jpg|center|link=]]</div>
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The important design choices regarding the biological work are motivated below.
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<p>Fig. 1 Yeast cell with the needed plasmids</p>
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<h3>Chassis</h3>
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<p>Before this light emitting cell display project could start, it was necessary to decide on a suitable chassis.  Candidates were E. coli and S. cerevisiae. Both are common model organisms that can be used for protein expression and are cheap to culture. To reach the goal of this project expression of <span class= "lightblue">voltage-gated calcium channel</span> is needed, which luckily is already the case for S. cerevisiae. Therefore, it was decided to use S. cerevisiae as our chassis throughout the project. More specifically, we used the <span class= "lightblue">INVSc1 yeast strain</span> which is compatible with the choice of vectors. Additional benefits when using yeast is that there are still only a <span class= "lightblue">few BioBricks<sup>TM</sup></span> available, which creates a greater opportunity to contribute to the Registry, but also the drawback of having less BioBricks<sup>TM</sup> available to ourselves. Another benefit is that, at the moment, the manipulation of yeast is <span class= "lightblue">less standardized</span> than it is for E. coli, which ultimately means that our research is <span class= "lightblue">more innovative</span>.</p>
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<h3>Plasmids and transformations</h3>
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<h4>Chassis</h4>
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<p>Since we choose yeast as the chassis we preferred to clone all required genes into <span class= "lightblue">shuttle vectors</span>. A shuttle vector is a special type of vector that can be propagated both in yeast and in bacteria. For our purposes we choose to use <span class= "lightblue">high copy-number vector</span> in order to achieve high concentration of GECO protein.</p>
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<p>Before this light emitting cell display project could start, it was necessary to decide on a suitable chassis.  Candidates were E. Coli and S. cerevisiae. Both are common model organisms that can be used for protein expression and are cheap to culture. To reach the goal of this project over expression of voltage-gated calcium channel was needed. As soon as it was found that CCH1-MID1, homologous to mammalian voltage-gated calcium channels, could be over expressed in S. cerevisiae, it was decided to use yeast as the chassis in our project.</p>
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<h4>Plasmids and transformations</h4>
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<p>The proteins CCH1 and MID1 together constitute a high-affinity Ca<sup>2+</sup>-channel. Both proteins are brought to expression on a <span class= "lightblue">low copy-number shuttle vector</span>. We are thankful to <span class= "lightblue">Hidetoshi Iida and Kazuko Iida</span> for their donation of the shuttle vectors pBCT-CCH1H and YCpT-MID1 which they constructed in earlier research.</p>
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<p>Since we choose yeast as our chassis we need to clone all required genes into shuttle vectors. A shuttle vector is a special type of vector that can be propagated both in yeast and in bacteria. Cloning can be done in fast growing E. coli while proteins can be expressed in yeast. There are low copy-number and high copy-number variants of shuttle vectors, the choice of which depends on the required amount of that protein in the cell. In this project we used three different shuttle vectors, each carrying one gene to be over expressed.</p>
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<p>The GECO proteins that were engineered in the group of <span class= "lightblue">Robert E. Campbell</span> (Zhao et al., 2011<sup>[2]</sup>) are available from Addgene.org, a non-profit plasmid sharing service. Via PCR we cloned the GECOs from the shipping plasmid into a pYES3 shuttle vector acquired from Invitrogen (Fig. 1).</p>
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<p>The proteins Cch1 and Mid1 together constitute a high-affinity Ca<sup>2+</sup>-channel. Both proteins are brought to over expression on a low copy-number shuttle vector. We are thankful to H. Iida & K. Iida for their donation of the shuttle vectors pBCT-CCH1H and YCpT-MID1 which they constructed in earlier research, and which were suited for use in our yeast.</p>
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<p>Transformation of S. cerevisiae cells can be done with a heat shock. To obtain a cell containing all the 3 different plasmids, <span class= "lightblue">3 consecutive transformations</span> are needed. Since we have 3 different GECOs, in the end we will have <span class= "lightblue">3 different variants</span>, all containing the CCH1-MID1 calcium channel but each with a different color of GECO.</p>
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<p>The GECO proteins that were engineered in the group of Robert E. Campbell (Zhao et al., 2011[2]) are available from Addgene.org, a non-profit plasmid sharing service. Via PCR we cloned the GECOs from the shipping plasmid into a pYES3 shuttle vector acquired from Invitrogen (Fig.1).</p>
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<p>The order in which plasmids are introduced can be varied. The route requiring the least transformations introduces first the CCH1 and MID1 proteins and finally the 3 different GECOs. Doing so, one can obtain all 3 variants by using 5 transformations, if the GECOs are first introduced the CCH1 and MID1 proteins, they need to be introduced separate to every GECO strain; this results in <span class= "lightblue">9 transformations in total</span>. Even though the second approach is more cumbersome, trying other routes in parallel provides a backup in case certain transformations fail.</p>
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<p>Transformation of S. cerevisiae cells can be done with a straightforward heat shock. To obtain a cell containing three different plasmids, three successive transformations are needed. Since we have three different GECOs, in the end we will have three different variants, all containing the CCH1-MID1 calcium channel but each with a different color of GECO.</p>
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<p>Selection of successful transformants is done by making use of <span class= "lightblue">auxotrophic markers</span>, a different one for each vector. The INVSc1 strain we use for transformations is auxotrophic in <span class= "lightblue">histidine, leucine, tryptophan and uracil</span>. These usually have to be added to the medium for the yeast to survive. The uptake of a vector however, will restore the ability of the cell to synthesize the missing amino acid or nucleic acid, and therefore it can survive in a selective medium.</p>
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<h3>Compatibility of yeast and device</h3>
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<p>The order in which plasmids are introduced can be varied. The route requiring the least transformations introduces CCH1 and MID1 first, then the GECOs. However, trying other routes in parallel provides a backup in case certain transformations fail.</p>
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<p>The genetically modified S. cerevisiae cells will be put to the test in our <span class= "lightblue">home-made</span> [[Team:TU-Eindhoven/LEC/Device|device]], designed for providing electrical stimuli to the yeast cells. These tests are done in a single bath filled with SC medium containing nutrients for our genetically modified yeast and additional free Ca<sup>2+</sup> ions. After <span class= "lightblue">electrical stimulation</span> of yeast at different positions in the device, optical signals will be expected to be visible to the naked eye. In a more <span class= "lightblue">sensitive analysis</span>, isolated protein will be characterized on a <span class="lightblue">spectrophotometer</span>.</p>
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<h3>GECOs</h3>
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<p>Selection of successful transformants is done by auxotrophic markers, one for each plasmid. The yeast strain we use for transformations is deficient in the synthesis of certain amino acids. These usually have to be added to the medium for the yeast to survive. The uptake of a plasmid however, will restore the ability of the cell to synthesize the missing amino acid and therefore survive in the medium.</p>
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<p>Real-time imaging of biochemical events inside living cells is important for understanding the molecular basis of physiological processes and diseases<html><a href="#ref_merkx" name="text_merkx"><sup>[3]</sup></a></html>. Genetically encoded sensors based on fluorescent proteins (FPs) are frequently used for <span class= "lightblue">molecular recognition</span>. In this iGEM project we use fluorescent proteins to provide the light in our display. More specifically we make use the so called GECOs.</p>
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<h3>Compatibility of yeast and device</h3>
 
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<p>The genetically modified S. Cerevisiae cells were put to the test in our home-made device, designed for providing electrical stimuli to the yeast cells. More about the device can be found in section ‘Device information’. These tests are done in a single bath filled with SC media containing nutrients for our genetically modified yeast and calcium. After electrical stimulation of yeast at different positions on the device, optical signals are seen by the naked eye and characterized by a plate reader.</p>
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[[File:Making_GECO.png|600px]]
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<p>Fig. 2 Cartoon representation of the calcium dependence</p>
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<h3>GECOs</h3>
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<p>Real-time imaging of biochemical events inside living cells is important for understanding the molecular basis of physiological processes and diseases<html><a href="#ref_merkx" name="text_merkx"><sup>[3]</sup></a></html>. Genetically encoded sensors based on fluorescent proteins (FPs) are frequently used for molecular recognition. In this iGEM project we use fluorescent proteins to provide the light in our display.</p>
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<p>A GECO is a protein which is fluorescent in the presence of Ca<sup>2+</sup><html><a href="#ref_zhao" name="text_zhao"><sup>[2]</sup></a></html> (Fig. 2). There are two important classes of <span class= "lightblue">genetically encoded Ca<sup>2+</sup> indicators</span>. One is called the Forster Resonance Energy Transfer (FRET)-based cameleon type<html><a href="#ref_miyawaki" name="text_miyawaki"><sup>[4]</sup></a></html> and the other one is called the single Green Fluorescent Protein (GFP) type<html><a href="#ref_nakai" name="text_nakai"><sup>[5]</sup></a></html>. The GECO protein belongs to the single GFP type. Research has shown that Ca<sup>2+</sup> indicators targeted to the E. coli periplasm can be shifted toward the Ca<sup>2+</sup>-free or Ca<sup>2+</sup>-bound states by <span class= "lightblue">manipulation of the intracellular Ca<sup>2+</sup> concentration</span><html><a href="#ref_zhao" name="text_zhao"><sup>[2]</sup></a></html>. Robert E. Campbell et al. named these Ca<sup>2+</sup> calcium sensors GECOs. <span class= "lightblue">R-GECO, G-GECO and B-GECO</span> emit red, green and blue light respectively, each with their own distinct excitation and emission spectrum (Fig. 3).</p>
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[[File:Fig.1. Emission Spectra GECO]]
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<p>A GECO is a protein which emits light in the presence of Ca<sup>2+</sup><html><a href="#ref_zhao" name="text_zhao"><sup>[2]</sup></a></html>. There are two important classes of genetically encoded Ca<sup>2+</sup> indicators. One is called the Forster Resonance Energy Transfer (FRET)-based cameleon type<html><a href="#ref_miyawaki" name="text_miyawaki"><sup>[4]</sup></a></html> and the other one is called the single Green Fluorescent Protein (GFP) type<html><a href="#ref_nakai" name="text_nakai"><sup>[5]</sup></a></html>. The GECO protein belongs to the single GFP type. Research has shown that Ca<sup>2+</sup> indicators targeted to the E.coli periplasm can be shifted toward the Ca<sup>2+</sup>-free or Ca<sup>2+</sup> -bound states by manipulation of the environmental Ca<sup>2+</sup> concentration<html><a href="#ref_zhao" name="text_zhao"><sup>[2]</sup></a></html>. Robert E. Campbell et al. named these Ca<sup>2+</sup> indicators GECOs. R-GECO, G-GECO and B-GECO emit red, green or blue light respectively, each with another emission and excitation spectrum (Fig. 1 and Fig2).</p>
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[[File:Excitation spectra GECO.png|300px]] [[File:Emission spectra GECO.png|300px]]
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<p>Fig. 3 Excitation and emission spectra of the GECO's</p>
<h3>References</h3>
<h3>References</h3>
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<li><a href="#text_Iida" name="ref_Iida">[1]</a> Iida, K. et al. 2007, Iida, K. et al. ''Essential, Completely Conserved Glycine Residue in the Domain III S2S3 Linker of Voltage-gated Calcium Channel α1 Subunits in Yeast and Mammals'' in ''Journal of Biological Chemistry'' August 31 2007, Vol. 282, issue 35 pp. 25659-25667, DOI: <a href="http://www.jbc.org/content/282/35/25659" target="_blank">10.1074/jbc.M703757200</a></li>
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<li><a href="#text_Iida" name="ref_Iida">[1]</a> K. Iida, et al., Essential, completely conserved glycine residue in the domain III S2S3 linker of voltage-gated calcium channel α1 subunits in yeast and mammals, Journal of Biological Chemistry 282: 25659-25667, (2007)</a></li>
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<li><a href="#text_zhao" name="ref_zhao">[2]</a> Zhao et al. 2011, Zhao, Y. et al. ''An Expanded Palette of Genetically Encoded Ca2+ Indicators'' in ''Science'' 30 September 2011, Vol. 333 no. 6051 pp. 1888-1891, DOI: <a href="http://www.sciencemag.org/content/333/6051/1888" target="_blank">10.1126/science.1208592</a></li>
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<li><a href="#text_zhao" name="ref_zhao">[2]</a> Y. Zhao, et al., An expanded palette of genetically encoded Ca2+ indicators, Science 333: 1888-1891, (2011)</a></li>
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<li><a href="#text_merkx" name="ref_merkx">[3]</a > Laurens Lindenburg and Maarten Merkx, ‘Colorful Calcium Sensors’, 2012</a></li>
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<li><a href="#text_merkx" name="ref_merkx">[3]</a > L. Lindenburg and M. Merkx, Colorful calcium sensors, (2012)</a></li>
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<li><a href="#text_miyawaki" name="ref_miyawaki">[4]</a> A. Miyawaki et al., Nature 338, 1997</a></li>
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<li><a href="#text_miyawaki" name="ref_miyawaki">[4]</a> A. Miyawaki, et al., Nature 338, (1997)</a></li>
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<li><a href="#text_nakai" name="ref_nakai">[5]</a> J. Nakai, M. Ohkura, K. Imoto, Nat. Biotechnol. (2001)</a></li>
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<li><a href="#text_nakai" name="ref_nakai">[5]</a> J. Nakai, M. Ohkura, K. Imoto, Nat. Biotechnol., (2001)</a></li>
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Latest revision as of 01:24, 27 September 2012