Team:Berkeley/Project/Localization

From 2012.igem.org

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By localizing fluorescent proteins to specific organelles, each cell can be given a "microscopic barcode", or MiCode. Below you can see a single sample MiCode. Each member of a library will get a unique MiCode, distinguishing it from the rest of the library and tying the MiCode phenotype to a specific genotype.
 
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To do this, we needed to decide which organelles to target and how to target them. We also had to optimize our choice of promoters so that one fluorescent protein signal was not too strong or too weak in comparison to the rest.
 
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We chose four organelles from a list of over ten candidates based on the following criteria: (1) there existed targeting sequences in the literature, and (2) this organelle was visibly distinct from the other chosen organelles. The following were our favorites from those we constructed!
 
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Our first main challenge of the summer was to determine which organelles we could easily and distinctly target in order to produce our MiCodes. We referenced the <a href="http://yeastgfp.yeastgenome.org/">YeastGFP database</a> of global analysis of protein localization studies in the budding yeast, <i>S.cerevisiae</i>, which was compiled by the O'Shea and Weissman labs at UCSF.  This database consists of microscopy analysis and characterization of GFP tagged to every ORF in yeast.  
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<img src="https://static.igem.org/mediawiki/2012/9/9e/YMRY045_nucleus.jpg" width="200">
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<p>Nucleus.
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<img  src="https://static.igem.org/mediawiki/2012/3/3c/YATJ012.jpg" width="200">
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<p>Cellular Periphery-We targeted GFP to the cell periphery using a signal sequence derived from the Ras protein, a signal protein located in the cell membrane that activates in response to extracellular signals for growth and differentiation.
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We found proteins that localized to various organelles, <a href="http://en.wikipedia.org/wiki/Pcr">PCR</a>'d them off of the genome, then tagged them with a fluorescent protein. After analyzing their specific localizations through microscopy, we narrowed our choices hoping to find consistent morphology and geometry.  
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<p>
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We chose four organelles from a list of over ten candidates based on the following criteria:
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<img src="https://static.igem.org/mediawiki/2012/8/83/YTC003_Actin.jpg" width="200">
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<ol>
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<p>Actin.
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<li>Existing targeting proteins or signal sequences in the literature.</li>
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<li>Visible distinction from the other chosen organelles.</li>
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</ol>
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<p>
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The following were our final four choices:
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<img src="https://static.igem.org/mediawiki/2012/c/c1/YMRY045_VM.jpg" width="200" >
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<p>Vacuolar Membrane
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<a href="https://static.igem.org/mediawiki/2012/9/9e/YMRY045_nucleus.jpg">
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<img src="https://static.igem.org/mediawiki/2012/b/ba/Nucleus.jpg" width="230"></a>
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<p><b>H2A2-Venus</b> The localizing protein is H2A2/YBL003C, a H2 histone protein that is essential for chromatin assembly. C-terminal protein fusion to H2A2 will localize the complex to the nucleus.
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<br>Registry Part:<a href="http://partsregistry.org/Part:BBa_K900002">BBa_K900002</a>
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<p><b>mKate-CIIC</b> The C-terminus CIIC signal sequence is derived from the Ras protein, a signal protein located in the plasma membrane that activates in response to extracellular signals for growth and differentiation.
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<br>Registry Part:<a href="http://partsregistry.org/Part:BBa_K900005">BBa_K900005</a>
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<a href="https://static.igem.org/mediawiki/2012/8/83/YTC003_Actin.jpg">
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<img src="https://static.igem.org/mediawiki/2012/4/46/Actin.jpg" width="230"></a>
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<p><b>ABP1-Venus </b> Actin-binding protein of the cortical actin cytoskeleton, important for activation of the Arp2/3 complex that plays a key role actin in cytoskeleton organization; phosphorylation within its PRR (Proline-Rich Region), mediated by Cdc28p and Pho85p, protects Abp1p from proteolysis mediated by its own PEST sequences. <br>Registry Part:<a href="http://partsregistry.org/Part:BBa_K900004">BBa_K900004</a>
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<a href="https://static.igem.org/mediawiki/2012/c/c1/YMRY045_VM.jpg">
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<img src="https://static.igem.org/mediawiki/2012/d/d1/VM.jpg" width="230" ></a>
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<p><b>ZRC1-mTurquoise</b> The localizing protein ZRC1/YMR243C is a vacuolar membrane zinc transporter. C-terminal protein fusion to ZRC1 will localize the complex to the vacuolar membrane.
 +
<br>Registry Part:<a href="http://partsregistry.org/Part:BBa_K900003">BBa_K900003</a>
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<p><i>*Protein characterizations provided by the <a href="http://yeastgfp.yeastgenome.org/">YeastGFP database</a>*
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We used RFP, GFP, and CFP and our four organelles that could be easily distinguished: the nucleus, vacuolar membrane, cellular periphery, and actin. Our number of MiCodes was represented by the function 2^x^y where x still represents the number of fluorescent proteins while y represents the number of organelles to which the fluorophores are targeted. With three fluorescent proteins and four organelles, the number of unique MiCodes is 4,096!
+
We used RFP, GFP, and CFP (which show up in red, green, and blue imaging channels, respectively) and our four organelles that could be easily distinguished: the nucleus, vacuolar membrane, plasma membrane, and actin. The size of our MiCode set can by calculated using the function (2^x)^y where x represents the number of imaging channels while y represents the number of organelles to which the fluorophores are targeted. With three imaging channels and four organelles, we can create <b>4,096</b> unique MiCode combinations!
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<br>
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<br>
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<p align="center">
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<img src="https://static.igem.org/mediawiki/2012/b/b2/Number_of_micodes.png" width="800" class="addborder">
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<p align="center">Our test MiCode set can accommodate up to 4,096 members!
 +
</p>
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<br>
-
<br><br>
+
<p>
 +
Although a MiCode set of up to 4,096 unique combinations is plenty for our proof of concept experiments, many experimental libraries would require a set size close to the magnitude of 10^6. With a few augmentations we can easily expand the available MiCode set to and even past 10^6 unique combinations.
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<img src="https://static.igem.org/mediawiki/2012/b/b2/Number_of_micodes.png" width="800">
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<p>
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Micode set size can be expanded by increasing the 3 criteria.
-
<br><br>
+
<ol>
 +
<li>
 +
<b>States:</b> A general binary system consists of genes expressing fluorescent proteins can either be on or off. Additionally, photosensitive fluorescent proteins that are activatable by UV light can be used as an extension into a ternary system in which the  states are: on, off , or photoactivatable (or PA for short).  
 +
<li>
 +
<b>Channels:</b> We currently utilize  three channels: red, green and blue. It is possible to use other fluorescent proteins that are viewable under other imaging channels without overlap.
 +
<li>
 +
<b>Organelles:</b> We optimized the use of four organelles (actin, plasma membrane, nucleus and vacuolar membrane) for our project but additional organelles can be utilized.
 +
</ol>
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</p>
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<br>
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<p align="center">
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<img src="https://static.igem.org/mediawiki/2012/6/64/Equation12.png" width="800">
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<p align="center">The more generalized equation for calculating the possible set of MiCodes.
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</p>
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<br>
 +
<p align="center">
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<img src="https://static.igem.org/mediawiki/2012/f/f6/Scale_up.png" width="400">
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<p align="center">By simply adding one more organelle and one more fluorescent protein that can be visualized under an additional channel, <br> the available MiCode set size <b>increases by 3 orders of magnitude</b>!
</p>
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When choosing promoters for our cassettes, we had to find promoters that would make each element of the MiCode visible under the microscope. Furthermore, the promoters would have to adjust for differences in protein abundances and consequently the relative brightness for each localization tag. A portion of our project was to decide on this optimum set of promoters.  
When choosing promoters for our cassettes, we had to find promoters that would make each element of the MiCode visible under the microscope. Furthermore, the promoters would have to adjust for differences in protein abundances and consequently the relative brightness for each localization tag. A portion of our project was to decide on this optimum set of promoters.  
 +
<br>
 +
<br>
 +
<p>
<p>
-
Out of the 7000+ promoters in the yeast genome, we decided to experiment with 5 promoters: pTEF1, pTDH3, pRPL18B, pRNR2, and pREV1 - listed from strongest to weakest. These were chosen based on the data from a paper from UCSF (Huh et al, 2003) in which every yeast genome open reading frame was tagged with GFP and observed for protein abundance. The 5 promoters listed above were simply 5 promoters that spanned a reasonable range in promoter strength and were promoters that Dueber lab was already familiar with.
+
Out of the 7000+ promoters in the yeast genome, we decided to experiment with 5 promoters: pTEF1, pTDH3, pRPL18B, pRNR2, and pREV1 - listed from strongest to weakest. These were chosen based on the data from a paper from UCSF <a href="http://www.nature.com/nature/journal/v425/n6959/full/nature02046.html">(Ghaemmaghami, et. al, 2003)</a> in which every yeast genome open reading frame was tagged with GFP and observed for protein abundance. The 5 promoters listed above were simply 5 promoters that spanned a reasonable range in promoter strength and were promoters that Dueber lab was already familiar with.
The promoter library we created allowed us to systematically compare all 5 promoters. Each yeast strain in the library expressed only one organelle localization tag, with one promoter, and we imaged them all under the same microscope settings. Two modes of image analysis were used for thorough comparison: one with the pixel brightness range set from 0-4095, and another with range 25-1000. The data can be seen <a href="Localization/PromoterLibrary"> here</a>.  
The promoter library we created allowed us to systematically compare all 5 promoters. Each yeast strain in the library expressed only one organelle localization tag, with one promoter, and we imaged them all under the same microscope settings. Two modes of image analysis were used for thorough comparison: one with the pixel brightness range set from 0-4095, and another with range 25-1000. The data can be seen <a href="Localization/PromoterLibrary"> here</a>.  
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To assay their potential use in our project, we characterized the strength of several registry yeast promoters. We wanted to compare their consistency of expression and relative fluorescence against our lab's strongest promoter, pTDH3. The promoters we used in our assay:
+
To assay their potential use in our project, we characterized the strength of several registry yeast promoters. We wanted to compare their consistency of expression and relative fluorescence, then quantify them against a standard promoter fluorescence. The promoters we used in our assay:
<ul>
<ul>
-
<li><a href="partsregistry.org/wiki/index.php?title=Part:BBa_I766557">pSTE5 (weak)</a>
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<li>pSTE5, a weak constitutive promoter for the yeast scaffold protein STE5. Registry Part: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I766557">BBa_I766557</a>.
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<li><a href="partsregistry.org/wiki/index.php?title=Part:BBa_I766555">pCYC1 (medium)</a>
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<li>pCYC1, a mid-level constitutive promoter for yeast Cytochrome c. Registry Part: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I766555">BBa_I766555</a>.
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<li><a href="partsregistry.org/wiki/index.php?title=Part:BBa_I766556">pADH1 (strong)</a>
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<li>pADH1, a strong constitutive promoter for yeast Alcohol Dehydrogenase. Registry Part: <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_I766556">BBa_I766556</a>.
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<li>pTDH3 (strongest)</a>
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<li>pTDH3, a strong constitutive promoter for the yeast glycerol synthesis protein GPD1. Registry Part: <a href="http://partsregistry.org/Part:BBa_K124002">BBa_K124002</a>.
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<b>Experimental Design</b>: We designed our promoters to express a fluorescent protein so that we could quickly measure bulk fluorescence via TECAN. To roughly test if downstream sequence affects expression, we cloned the promoter in front of two different fluorescent proteins, yellow fluorescent Venus and red fluorescent mKate. The device was cloned on a backbone with Leu2 marker and Cen6 origin of replication.  
+
<b>Experimental Design</b>: We designed our promoters to express a fluorescent protein so that we could quickly measure bulk fluorescence via TECAN. To test if downstream sequence affected expression, we cloned the promoter in front of two different fluorescent proteins, yellow fluorescent Venus and red fluorescent mKate in different strains. If expression was sequence-independent, we expected similar fluorescence intensity from both channels. The device was cloned on a backbone with Leu2 marker and Cen6 origin of replication and transformed into S228C <i>S. cerevisiae</i>. Ideally, we wanted the calibration curve to span several orders of magnitude and exhibit a linear relationship, which was true for all the promoters we characterized except for pCYC1.
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After
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We collected data for 8 samples per strain. the averaged <b>data</b> that we acquired is shown below:
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<img src="https://static.igem.org/mediawiki/2012/0/0f/IGEM_promoter_characterization.png" width="600" class="addborder">
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<p align="center">Calibration curve of fluorescence intensity. Error bars are +/- one standard deviation.
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</p>
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<table align="center">
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    <th>Promoter</th>
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    <th>RFP/OD</th>
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    <th>YFP/OD</th>
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    <td>background</td>
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    <td>55 ± 10</td>
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    <td>36 ± 2</td>
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    <td>pSTE5</td>
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    <td>126 ± 10</td>
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    <td>216 ± 20</td>
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    <td>pCYC1</td>
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    <td>189 ± 30</td>
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    <td>928 ± 30</td>
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    <td>pADH1</td>
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    <td>1170 ± 90</td>
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    <td>4360 ± 100</td>
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  </tr>
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  <tr>
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    <td>pTDH3</td>
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    <td>22164 ± 700</td>
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    <td>62531 ± 2000</td>
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Latest revision as of 05:00, 22 October 2012

header
iGEM Berkeley iGEMBerkeley iGEMBerkeley

Mercury


Our first main challenge of the summer was to determine which organelles we could easily and distinctly target in order to produce our MiCodes. We referenced the YeastGFP database of global analysis of protein localization studies in the budding yeast, S.cerevisiae, which was compiled by the O'Shea and Weissman labs at UCSF. This database consists of microscopy analysis and characterization of GFP tagged to every ORF in yeast.


We found proteins that localized to various organelles, PCR'd them off of the genome, then tagged them with a fluorescent protein. After analyzing their specific localizations through microscopy, we narrowed our choices hoping to find consistent morphology and geometry.


We chose four organelles from a list of over ten candidates based on the following criteria:

  1. Existing targeting proteins or signal sequences in the literature.
  2. Visible distinction from the other chosen organelles.

The following were our final four choices:

H2A2-Venus The localizing protein is H2A2/YBL003C, a H2 histone protein that is essential for chromatin assembly. C-terminal protein fusion to H2A2 will localize the complex to the nucleus.
Registry Part:BBa_K900002

mKate-CIIC The C-terminus CIIC signal sequence is derived from the Ras protein, a signal protein located in the plasma membrane that activates in response to extracellular signals for growth and differentiation.
Registry Part:BBa_K900005

ABP1-Venus Actin-binding protein of the cortical actin cytoskeleton, important for activation of the Arp2/3 complex that plays a key role actin in cytoskeleton organization; phosphorylation within its PRR (Proline-Rich Region), mediated by Cdc28p and Pho85p, protects Abp1p from proteolysis mediated by its own PEST sequences.
Registry Part:BBa_K900004

ZRC1-mTurquoise The localizing protein ZRC1/YMR243C is a vacuolar membrane zinc transporter. C-terminal protein fusion to ZRC1 will localize the complex to the vacuolar membrane.
Registry Part:BBa_K900003


*Protein characterizations provided by the YeastGFP database*



We used RFP, GFP, and CFP (which show up in red, green, and blue imaging channels, respectively) and our four organelles that could be easily distinguished: the nucleus, vacuolar membrane, plasma membrane, and actin. The size of our MiCode set can by calculated using the function (2^x)^y where x represents the number of imaging channels while y represents the number of organelles to which the fluorophores are targeted. With three imaging channels and four organelles, we can create 4,096 unique MiCode combinations!

Our test MiCode set can accommodate up to 4,096 members!


Although a MiCode set of up to 4,096 unique combinations is plenty for our proof of concept experiments, many experimental libraries would require a set size close to the magnitude of 10^6. With a few augmentations we can easily expand the available MiCode set to and even past 10^6 unique combinations.


Micode set size can be expanded by increasing the 3 criteria.

  1. States: A general binary system consists of genes expressing fluorescent proteins can either be on or off. Additionally, photosensitive fluorescent proteins that are activatable by UV light can be used as an extension into a ternary system in which the states are: on, off , or photoactivatable (or PA for short).
  2. Channels: We currently utilize three channels: red, green and blue. It is possible to use other fluorescent proteins that are viewable under other imaging channels without overlap.
  3. Organelles: We optimized the use of four organelles (actin, plasma membrane, nucleus and vacuolar membrane) for our project but additional organelles can be utilized.


The more generalized equation for calculating the possible set of MiCodes.


By simply adding one more organelle and one more fluorescent protein that can be visualized under an additional channel,
the available MiCode set size increases by 3 orders of magnitude!

When choosing promoters for our cassettes, we had to find promoters that would make each element of the MiCode visible under the microscope. Furthermore, the promoters would have to adjust for differences in protein abundances and consequently the relative brightness for each localization tag. A portion of our project was to decide on this optimum set of promoters.

Out of the 7000+ promoters in the yeast genome, we decided to experiment with 5 promoters: pTEF1, pTDH3, pRPL18B, pRNR2, and pREV1 - listed from strongest to weakest. These were chosen based on the data from a paper from UCSF (Ghaemmaghami, et. al, 2003) in which every yeast genome open reading frame was tagged with GFP and observed for protein abundance. The 5 promoters listed above were simply 5 promoters that spanned a reasonable range in promoter strength and were promoters that Dueber lab was already familiar with. The promoter library we created allowed us to systematically compare all 5 promoters. Each yeast strain in the library expressed only one organelle localization tag, with one promoter, and we imaged them all under the same microscope settings. Two modes of image analysis were used for thorough comparison: one with the pixel brightness range set from 0-4095, and another with range 25-1000. The data can be seen here.


To assay their potential use in our project, we characterized the strength of several registry yeast promoters. We wanted to compare their consistency of expression and relative fluorescence, then quantify them against a standard promoter fluorescence. The promoters we used in our assay:

  • pSTE5, a weak constitutive promoter for the yeast scaffold protein STE5. Registry Part: BBa_I766557.
  • pCYC1, a mid-level constitutive promoter for yeast Cytochrome c. Registry Part: BBa_I766555.
  • pADH1, a strong constitutive promoter for yeast Alcohol Dehydrogenase. Registry Part: BBa_I766556.
  • pTDH3, a strong constitutive promoter for the yeast glycerol synthesis protein GPD1. Registry Part: BBa_K124002.

Experimental Design: We designed our promoters to express a fluorescent protein so that we could quickly measure bulk fluorescence via TECAN. To test if downstream sequence affected expression, we cloned the promoter in front of two different fluorescent proteins, yellow fluorescent Venus and red fluorescent mKate in different strains. If expression was sequence-independent, we expected similar fluorescence intensity from both channels. The device was cloned on a backbone with Leu2 marker and Cen6 origin of replication and transformed into S228C S. cerevisiae. Ideally, we wanted the calibration curve to span several orders of magnitude and exhibit a linear relationship, which was true for all the promoters we characterized except for pCYC1.

We collected data for 8 samples per strain. the averaged data that we acquired is shown below:

Calibration curve of fluorescence intensity. Error bars are +/- one standard deviation.


Promoter RFP/OD YFP/OD
background 55 ± 10 36 ± 2
pSTE5 126 ± 10 216 ± 20
pCYC1 189 ± 30 928 ± 30
pADH1 1170 ± 90 4360 ± 100
pTDH3 22164 ± 700 62531 ± 2000




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