Difference between revisions of "Team:UC San Diego/Module"

 
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<h3>Description</h3>
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                  <h1>Module</h1>
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All coding sequences have been codon optimized for expression in the yeast <i>Saccharomyces Cerevisiae</i>. By expressing this system in a simple eukaryotic organism, we pave the way for expression of the Lux system in higher level organisms, increasing its utility as an <i>in vivo</i> reporter that does not rely on the addition of a substrate.
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To identify the rate-limiting enzyme of the Lux system in yeast, we have expressed three permutations of the <i>lux</i>CDE genes from <i>Photobacterium Phosphoreum</i> in combination with with the <i>frp</i> gene from <i>Vibrio Harveyi</i> and an “enhanced” LuxAB fusion protein<sup>1,2</sup>.
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<a href="https://static.igem.org/mediawiki/2015/6/61/UCSD_plasmidcont.jpg" data-lightbox="labplasmid" data-title="The genes <i>lux</i>AB and <i>frp</i> are expressed under the HXT7 promoter, while the <i>lux</i>CDE genes are expressed under the TEF1 promoter. Use of this bidirectional promoter system as opposed to the expression of the genes under a single promoter facilitates a modular approach to plasmid design, allowing us to easily test multiple stoichiometric combinations."><center><img width="600" src="https://static.igem.org/mediawiki/2015/6/61/UCSD_plasmidcont.jpg"></center></a>
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<div class="caption"><i>The genes </i>lux<i>AB and </i>frp<i> are expressed under the HXT7 promoter, while the </i>lux<i>CDE genes are expressed under the TEF1 promoter. Use of this bidirectional promoter system as opposed to the expression of the genes under a single promoter facilitates a modular approach to plasmid design, allowing us to easily test multiple stoichiometric combinations.</i></div>
  
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<p>In our luminescent system, we coexpressed the luxCDE genes from Photobacterium Phosphoreum with the frp gene from Vibrio Harveyi and a modified LuxAB gene.<br><br>
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Because of the detailed <i>in vitro</i> characterization of its fatty acid reductase complex structure and function, we chose to use the genes from <i>Photobacterium Phosphoreum</i>. A configuration with a single copy of each gene will be used to establish a baseline level of luminescence. Each other permutation of the <i>lux</i>CDE construct will involve the overproduction of a different Lux C, D, or E enzyme. By comparing luminescence after overproduction for each enzyme to this baseline level, we easily can identify the rate-limiting enzyme and associated rate-limiting step.  
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[Plasmid Diagram]<br><br>
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To accurately control the relative amounts of each subunit of the complex present in the cell, the genes in our system are separated from each other by P2A linker sequences. These linkers are peptide sequences that cause the ribosome to skip the formation of a peptide bond between its terminal glycine and proline residues<sup>3</sup>. As a result, the coded proteins are expressed in precise ratios, allowing for the stoichiometric control of multiple proteins from a single strand of mRNA.
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To identify the rate limiting enzyme in the synthesis reaction, we have chosen to create three permutations of these genes for expression. By placing a 2A linker sequence between each gene, we can allow for their stoichiometric expression upon translation.<br><br>
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<center><a href="https://static.igem.org/mediawiki/2015/8/8b/UCSD_Permutations2.png" data-lightbox="permutations" data-title="Through the use of 2A linkers to control enzyme stoichiometry, we can express these three genes in a precise 1:1:2 ratio."><img width="700" src="https://static.igem.org/mediawiki/2015/8/8b/UCSD_Permutations2.png"></a></center>
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Some kind of “About 2A sequences” blurb: 2A linkers are peptide sequences that cause the ribosome to skip the formation of a peptide bond between its terminal glycine and proline residues. This allows for the stoichiometric coexpression of multiple proteins from a single strand of mRNA. We used the P2A linker, which has the following sequence: (GSG) A T N F S L L K Q A G D V E E N P G P.<br><br>
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<div class="caption"><center><i>Through the use of 2A linkers to control enzyme stoichiometry, we can express these three genes in a precise 1:1:2 ratio.</i></center></div>
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[Diagram of Permutations w/ elaborating caption: Because the 2A linkers allow us to control enzyme stoichiometry, we can express these three genes in a precise 1:1:2 ratio. ]
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The <i>frp</i> gene from <i>Vibrio Harveyi</i> allows for the regeneration of FMNH<sub>2</sub>, an essential cofactor for luciferase activity. Previous attempts to express the Lux system in yeast in the absence of FMNH<sub>2</sub> resulted in weak, unstable light output, which stabilized and increased after addition of the gene<sup>2</sup>.
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Instead of using the <i>lux</i>AB genes native to <i>Photobacterium Phosphoreum</i>, we have opted to use an engineered, monomeric luciferase designed for greater stability and luminescent yield<sup>1</sup>. Though the expression of unmodified luciferase has been shown to be sufficient for light-generation, the engineered luciferase has been shown to increase the reaction’s light output, generating a stronger signal. This also minimizes the possibility that LuxAB is rate limiting, masking the rate-limiting steps in the CDE reactions.
 
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<h3>Plasmid Maps</h3>
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<center><img src="https://static.igem.org/mediawiki/2015/e/e2/UCSD_plasmidluxC.jpg" width='600'><br><br>
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<p>The frp gene from Vibrio Harveyi allows for the regeneration of FMNH2 for the light-generating reaction. In eukaryotic cells, FMNH2 is not replenished at a rate sufficient for strong, consistent luminescence, necessitating the introduction of an enzyme to facilitate the process. <br><br>
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<img src="https://static.igem.org/mediawiki/2015/b/bd/UCSD_plasmidluxD.jpg" width='600'><br><br>
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<img src="https://static.igem.org/mediawiki/2015/5/5d/UCSD_plasmidluxE.jpg" width='600'></center>
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Instead of using the LuxAB genes native to Photobacterium Phosphoreum, we have opted to use an engineered, monomeric luciferase designed for greater stability and luminescent yield. Though the expression of unmodified luciferase has been shown to be sufficient for light-generation, the engineered luciferase has been shown to increase the reaction’s light output, generating a stronger signal. <br><br>
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<h3>References</h3>
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All coding sequences have been codon optimized for expression in yeast. </p>
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<center>CITATIONS</center>
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[1] Cui B, Zhang L, Song Y, Wei J, Li C, et al. (2014) Engineering an Enhanced, Thermostable, Monomeric Bacterial Luciferase Gene As a Reporter in Plant Protoplasts. PLoS ONE 9(10): e107885. doi:10.1371/journal.pone.0107885
 
[1] Cui B, Zhang L, Song Y, Wei J, Li C, et al. (2014) Engineering an Enhanced, Thermostable, Monomeric Bacterial Luciferase Gene As a Reporter in Plant Protoplasts. PLoS ONE 9(10): e107885. doi:10.1371/journal.pone.0107885
 
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[2] Gupta, R. K., Patterson, S. S., Ripp, S., Simpson, M. L. and Sayler, G. S. (2003), Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. FEMS Yeast Research, 4: 305–313. doi: 10.1016/S1567-1356(03)00174-0
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[2] Gupta, R. K., Patterson, S. S., Ripp, S., Simpson, M. L. and Sayler, G. S. (2003), Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. FEMS Yeast Research,4: 305–313. doi: 10.1016/S1567-1356(03)00174-0
 
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[3]Szymczak-Workman AL, Vignali KM, Vignali DA. Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harbor Protoc. 2012(2): 199–204. doi: 10.1101/pdb.ip067876
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[3] Szymczak-Workman AL, Vignali KM, Vignali DA. Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harbor Protoc. 2012(2): 199–204. doi: 10.1101/pdb.ip067876
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Latest revision as of 21:14, 20 November 2015

Description

All coding sequences have been codon optimized for expression in the yeast Saccharomyces Cerevisiae. By expressing this system in a simple eukaryotic organism, we pave the way for expression of the Lux system in higher level organisms, increasing its utility as an in vivo reporter that does not rely on the addition of a substrate.

To identify the rate-limiting enzyme of the Lux system in yeast, we have expressed three permutations of the luxCDE genes from Photobacterium Phosphoreum in combination with with the frp gene from Vibrio Harveyi and an “enhanced” LuxAB fusion protein1,2.


The genes luxAB and frp are expressed under the HXT7 promoter, while the luxCDE genes are expressed under the TEF1 promoter. Use of this bidirectional promoter system as opposed to the expression of the genes under a single promoter facilitates a modular approach to plasmid design, allowing us to easily test multiple stoichiometric combinations.

Because of the detailed in vitro characterization of its fatty acid reductase complex structure and function, we chose to use the genes from Photobacterium Phosphoreum. A configuration with a single copy of each gene will be used to establish a baseline level of luminescence. Each other permutation of the luxCDE construct will involve the overproduction of a different Lux C, D, or E enzyme. By comparing luminescence after overproduction for each enzyme to this baseline level, we easily can identify the rate-limiting enzyme and associated rate-limiting step.

To accurately control the relative amounts of each subunit of the complex present in the cell, the genes in our system are separated from each other by P2A linker sequences. These linkers are peptide sequences that cause the ribosome to skip the formation of a peptide bond between its terminal glycine and proline residues3. As a result, the coded proteins are expressed in precise ratios, allowing for the stoichiometric control of multiple proteins from a single strand of mRNA.


Through the use of 2A linkers to control enzyme stoichiometry, we can express these three genes in a precise 1:1:2 ratio.

The frp gene from Vibrio Harveyi allows for the regeneration of FMNH2, an essential cofactor for luciferase activity. Previous attempts to express the Lux system in yeast in the absence of FMNH2 resulted in weak, unstable light output, which stabilized and increased after addition of the gene2.

Instead of using the luxAB genes native to Photobacterium Phosphoreum, we have opted to use an engineered, monomeric luciferase designed for greater stability and luminescent yield1. Though the expression of unmodified luciferase has been shown to be sufficient for light-generation, the engineered luciferase has been shown to increase the reaction’s light output, generating a stronger signal. This also minimizes the possibility that LuxAB is rate limiting, masking the rate-limiting steps in the CDE reactions.

Plasmid Maps





References

[1] Cui B, Zhang L, Song Y, Wei J, Li C, et al. (2014) Engineering an Enhanced, Thermostable, Monomeric Bacterial Luciferase Gene As a Reporter in Plant Protoplasts. PLoS ONE 9(10): e107885. doi:10.1371/journal.pone.0107885
[2] Gupta, R. K., Patterson, S. S., Ripp, S., Simpson, M. L. and Sayler, G. S. (2003), Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. FEMS Yeast Research,4: 305–313. doi: 10.1016/S1567-1356(03)00174-0
[3] Szymczak-Workman AL, Vignali KM, Vignali DA. Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harbor Protoc. 2012(2): 199–204. doi: 10.1101/pdb.ip067876