Team:TU-Eindhoven/LEC/Lab

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<p>The 3 GECOs have been expressed in E. coli BL21 to yield a large amount of the proteins for characterization. Cells were <span class = "lightblue">cultured</span> in large Erlenmeyer flasks. The <span class = "lightblue">results were visually impressive</span>: After lysis the flasks turned vibrantly red and green respectively. The blue GECO however looked much the same as the green GECO.</p>
<p>The 3 GECOs have been expressed in E. coli BL21 to yield a large amount of the proteins for characterization. Cells were <span class = "lightblue">cultured</span> in large Erlenmeyer flasks. The <span class = "lightblue">results were visually impressive</span>: After lysis the flasks turned vibrantly red and green respectively. The blue GECO however looked much the same as the green GECO.</p>
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<p>More information about the GECOs can be found at [[Team:TU-Eindhoven/Parts|Our BioBricks<sup>TM</sup>]].</p>
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<p>More information about the GECOs can be found at [[Team:TU-Eindhoven/Parts|our BioBricks<sup>TM</sup>]].</p>
<h3>Yeast transformation</h3>
<h3>Yeast transformation</h3>
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<p>A weekly item on our schedule was <span class = "lightblue">yeast transformation</span>. It takes about a week to complete a transformation, that is, to add one plasmid to an existing strain variant. Because success is not guaranteed we choose to introduce plasmids in various orders in parallel. This resulted in <span class = "lightblue">many variants</span> that we assigned a unique number for convenience. The same number was used on plates, cultures and cryostocks. In total <span class = "lightblue">49 variants</span> were made.</p>
<p>A weekly item on our schedule was <span class = "lightblue">yeast transformation</span>. It takes about a week to complete a transformation, that is, to add one plasmid to an existing strain variant. Because success is not guaranteed we choose to introduce plasmids in various orders in parallel. This resulted in <span class = "lightblue">many variants</span> that we assigned a unique number for convenience. The same number was used on plates, cultures and cryostocks. In total <span class = "lightblue">49 variants</span> were made.</p>
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[[File:Yeast_transformation.jpg|300px|left|link=|thumb]]<p>To reach the aim of this project and prepare a multi-colored screen, three kind of cells, each with a different color, are needed. Therefore, one single plasmid strain would have to consist of <span class = "lightblue">both calcium channel parts MID1 and CCH1 sequences and one of the fluorescent GECO protein sequence</span>. Each of these DNA sequences, coding for the three different parts, is transformed into a yeast plasmid (InvSC1). Moreover, each of these sequences contain a small sequence coding for an amino acid, causing the yeast strain to be able to synthesize this specific amino acid. The synthesis of this specific amino acid will allow strain selection by retaining this amino acid from the culture media. Cells containing the  coding CCH1, MID1 and GECO sequence are able to <span class = "lightblue">synthesize leucine, uracil and tryptophan</span> respectively. </p>
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[[File:Yeast_transformation.jpg|300px|right|link=]]<p>To reach the aim of this project and prepare a multi-colored screen, three kind of cells, each with a different color, are needed. Therefore, one single plasmid strain would have to consist of <span class = "lightblue">both calcium channel parts MID1 and CCH1 sequences and one of the fluorescent GECO protein sequence</span>. Each of these DNA sequences, coding for the three different parts, is transformed into a yeast plasmid (InvSC1). Moreover, each of these sequences contain a small sequence coding for an amino acid, causing the yeast strain to be able to synthesize this specific amino acid. The synthesis of this specific amino acid will allow strain selection by retaining this amino acid from the culture media. Cells containing the  coding CCH1, MID1 and GECO sequence are able to <span class = "lightblue">synthesize leucine, uracil and tryptophan</span> respectively. </p>
<p>Looking at Scheme 1, which represents the <span class = "lightblue">transformations</span> needed to come to the desired yeast strain, it seems and is logical to start with the MID1 or CCH1 vectors. By doing so, less transformations can be performed due to the transformation intensive introduction of the three different GECO proteins at last. To make sure that the transformations would lead to the desired strain, it was decided to start the first step with transforming <span class = "lightblue">all possibilities</span>. When step 1 was transformed successfully, visualized by the arrows in Scheme 1, the transformation of step 2 was performed and so on.</p>
<p>Looking at Scheme 1, which represents the <span class = "lightblue">transformations</span> needed to come to the desired yeast strain, it seems and is logical to start with the MID1 or CCH1 vectors. By doing so, less transformations can be performed due to the transformation intensive introduction of the three different GECO proteins at last. To make sure that the transformations would lead to the desired strain, it was decided to start the first step with transforming <span class = "lightblue">all possibilities</span>. When step 1 was transformed successfully, visualized by the arrows in Scheme 1, the transformation of step 2 was performed and so on.</p>
[[File:Transformation chart.png|710px|link=]]
[[File:Transformation chart.png|710px|link=]]
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<p>Fluorescent proteins like our GECOs can be detected by <span class = "lightblue">spectrophotometry</span> if the intensity is insufficient to be observed by the naked eye. If many samples have to be measured these can be put onto a 96-wells plate and measured all at once in a special type of spectrophotometer called a <span class = "lightblue">plate reader</span>. A plate reader is also convenient for use with small sample volumes, which was beneficial in our case, since it allowed us to concentrate the yeast from a large culture volume into a small volume through centrifugation and obtain a better measurement.</p>
<p>Fluorescent proteins like our GECOs can be detected by <span class = "lightblue">spectrophotometry</span> if the intensity is insufficient to be observed by the naked eye. If many samples have to be measured these can be put onto a 96-wells plate and measured all at once in a special type of spectrophotometer called a <span class = "lightblue">plate reader</span>. A plate reader is also convenient for use with small sample volumes, which was beneficial in our case, since it allowed us to concentrate the yeast from a large culture volume into a small volume through centrifugation and obtain a better measurement.</p>
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[[File:GECO_on_plate_reader_96_wells.jpg|left|300px|link=|thumb|Samples of yeast on a 96-wells plate.]]<p>The expression of GECOs in yeast did <span class = "lightblue">not yield obvious fluorescence</span> when [[Team:TU-Eindhoven/LEC/Device#practice|stimulated in the device]]. To check if the protein was actually present in our cells, we made spectrograms of cells concentrated to 1.25 x 10<sup>8</sup> cells/ml, which amounts to an OD<sub>600</sub> of approximately 125.</p>
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[[File:GECO_on_plate_reader_96_wells.jpg|left|300px|link=|thumb|Samples of yeast on a 96-wells plate.]]<p>The expression of GECOs in yeast did <span class = "lightblue">not yield obvious fluorescence</span> when [[Team:TU-Eindhoven/LEC/Device|stimulated in the device]]. To check if the protein was actually present in our cells, we made spectrograms of cells concentrated to 1.25 x 10<sup>8</sup> cells/ml, which amounts to an OD<sub>600</sub> of approximately 125.</p>
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<p>There is a <span class = "lightblue">clear difference</span> in spectra between R-GECO and G-GECO, which means they can be <span class = "lightblue">used together</span> in a multi-colored screen without disturbing each others emission. The difference between G-GECO and B-GECO is very small, perhaps another excitation wavelength could better separate the emission spectra and make them better suited for simultaneous use in the same screen.</p>
<p>There is a <span class = "lightblue">clear difference</span> in spectra between R-GECO and G-GECO, which means they can be <span class = "lightblue">used together</span> in a multi-colored screen without disturbing each others emission. The difference between G-GECO and B-GECO is very small, perhaps another excitation wavelength could better separate the emission spectra and make them better suited for simultaneous use in the same screen.</p>
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<p>The following non-BioBrick plasmids have been constructed:</p>
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Latest revision as of 02:17, 27 September 2012