Difference between revisions of "Part:BBa K1692008"

 
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<h2>Overview</h2>
 
<h2>Overview</h2>
  
This operon is a composite of three enzymes in the following order: [https://parts.igem.org/Part:BBa_K1692002 FDC (BBa_K1692002)], [https://parts.igem.org/Part:BBa_K1692007 UbiX (BBa_K1692007)], and [https://parts.igem.org/Part:BBa_K1692004 PAL (BBa_K1692004)].
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<p>This operon is a composite of the three enzymes involved in styrene synthesis in the following order: [https://parts.igem.org/Part:BBa_K1692002 FDC (BBa_K1692002)], [https://parts.igem.org/Part:BBa_K1692007 UbiX (BBa_K1692007)], and [https://parts.igem.org/Part:BBa_K1692004 PAL (BBa_K1692004)].The entire operon is controlled via an inducible T7 promoter. Each protein-coding sequence is preceded by a ribosome binding site and followed by a FLAG-tag peptide, enabling easy and efficient extraction</p>
  
PAL converts phenylalanine to cinnamic acid.
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[[File:Screen Shot 2015-09-13 at 7.57.36 PM.png|thumbnail|center|700px|<b>Styrene Synthesis Operon</b>]]<br><br>
FDC converts cinnamic acid to styrene.
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UbiX modifies flavin mononucleotide to produce a cofactor that is required for FDC activity.
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The entire operon is controlled via an inducible T7 promoter. Each protein-coding sequence is preceded by a ribosome binding site and followed by a FLAG-tag peptide, enabling easy and efficient extraction.
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[[File:SB2015_styrene_pathway.png|thumbnail|center|500px|<b>Styrene synthesis pathway</b>  The enzymes of interest are phenylalanine ammonia lyase (PAL), ferulic acid decarboxylase (FDC), and a flavin prenyltransferase involved in ubiquinone biosynthesis called UbiX. PAL catalyzes the conversion of phenylalanine to trans-cinnamic acid, while FDC catalyzes the conversion of trans-cinnamic acid to styrene [2]. Recently, it has been discovered that a cofactor is required to activate FDC. This cofactor is a product of the reaction between dimethylallyl monophosphate (DMAP) and flavin mononucleotide (FMN), which is catalyzed by the enzyme UbiX [3].]]
 
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[[File:SB2015_styrene_pathway.png|thumbnail|center|500px|<b>Styrene synthesis pathway</b>  The enzymes of interest are phenylalanine ammonia lyase (PAL), ferulic acid decarboxylase (FDC), and a flavin prenyltransferase involved in ubiquinone biosynthesis called UbiX. PAL catalyzes the conversion of phenylalanine to trans-cinnamic acid, while FDC catalyzes the conversion of trans-cinnamic acid to styrene [1]. Recently, it has been discovered that a cofactor is required to activate FDC. This cofactor is a product of the reaction between dimethylallyl monophosphate (DMAP) and flavin mononucleotide (FMN), which is catalyzed by the enzyme UbiX [2].]]<br><br>
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<h2>Experiments and Results</h2>
 
<h2>Experiments and Results</h2>
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The decision to place FDC first, PAL last, and UbiX in between was based on results from our mathematical model. Specifically, our model suggested that FDC exerts greatest control on the flux of biomaterials through the pathway; in other words, changes in FDC concentration result in the greatest changes in final styrene production. We combined this finding with the fact that earlier genes in an operon are expressed at higher levels [1]. Taken together, these results implied that we should place FDC first.  Additionally, since UbiX’s cofactor product is essential for FDC activity, we decided to place UbiX immediately after FDC.
  
After obtaining our synthesized gene, we needed to insert it into the standard pSB1C3 backbone so we could transform it and submit as a biobrick.  To do this we digested our linear gene and standard iGEM RFP plasmid ([https://parts.igem.org/Part:BBa_J04450 BBa_J04450]) with a combination of EcoRI and SpeI or PstI restriction enzymes.  We then ligated with T4 ligase and transformed into NEB 5-alpha competent E. coli cells. Now that we had our gene in a plasmid with a promoter and RBS we transformed it into T7 expressing NEB E. coli.  We grew up large cultures, which we initiated T7 polymerase gene expression by adding IPTG to our cultures.  Because all of our synthesized genes had a FLAG tag at the end of their sequence, we were able to purify our proteins from the cell lysate.  To do this we used the Anti-FLAG Tag protein purification method.  We then used a BCA protein assay to determine the concentrations of our purified proteins.  Finally we ran all three of our purified enzymes on SDS PAGE with a Mark 12 protein ladder to verify that our proteins were the correct molecular weight, which they were.
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[[File:SB2015 StyreneEnzymeInfluence.png|thumbnail|center|700px|<b></b>]]<br><br>
 
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[[File:SB2015_styrene_enzymes_SDS_PAGE.png|thumbnail|center|700px|This is a <b>SDS PAGE gel</b> with purified PAL, FDC and UbiX protein.  We ran a Mark 12 protein ladder to verify that our proteins were the correct molecular weight.]]<br><br>
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The decision to place FDC first, PAL last, and UbiX in between was based on results from our mathematical model. Specifically, our model suggested that FDC exerts greatest control on the flux of biomaterials through the pathway; in other words, changes in FDC concentration result in the greatest changes in final styrene production. We combined this finding with the fact that earlier genes in an operon are expressed at higher levels. Taken together, these results implied that we should place FDC first.
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<p>Once we determined the gene order for our FDC-UbiX-PAL operon, we needed to make it. To do this we used the New England Biolabs Gibson Assembly kit.  We designed eight primers according to a primer design protocol (also from NEB) for FDC, UbiX, PAL and RFP plasmids. We then ran a PCR extension on all four of the resulting segments. We ran these four products on a gel and gel extracted the bands that appeared at the desired migration distances. We used this purified product for the Gibson assembly and transformed our resultant plasmid into NEB 5-alpha competent cells (NEB).  We ran a colony PCR and found three colonies with the expected band length of over 4,000 base pairs.  We grew up liquid cultures of these three colonies, miniprepped, and ran sequencing using five internal primers designed specifically for the combo plasmid. We found that two of the three colonies had the correct sequence! </p>
  
Additionally, since UbiX’s cofactor product is essential for FDC activity, we decided to place UbiX immediately after FDC.
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<p>We transformed the combo plasmid with FDC-UbiX-PAL operon (sequence confirmed) into T7 expressing competent cells (T7 Express, NEB) and performed a western blot using FITC-conjugated anti-flag antibody (Sigma) to confirm that all three genes were actually expressing the recombinant proteins with expected sizes. </p>
  
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[[File:Western-SB2015.jpg|thumbnail|center|500px|<b>Western blot analysis of <i>in vivo</i> expressed FDC-PAL-Ubix proteins</b>]]
  
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<p>Confirming the clear over-expression of FDC over PAL protein from the FDC-UbiX-PAL operon <i>in vivo</i>, we are currently performing GC-MS analysis to see if the cells are fully capable of producing styrene at detectable and useful levels.</p>
 
<h2>Reference</h2>
 
<h2>Reference</h2>
[1] Mckenna, Rebekah, Luis Moya, Matthew Mcdaniel, and David R. Nielsen. "Comparing in Situ Removal Strategies for Improving Styrene Bioproduction." Bioprocess Biosyst Eng Bioprocess and Biosystems Engineering (2014): 165-74. Print.  
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<p>[1] Lim, H. N., Y. Lee, and R. Hussein. "Fundamental Relationship between Operon Organization and Gene Expression." Proceedings of the National Academy of Sciences (2011): 10626-0631. Print. </p>
<p>[2] White, Mark D., Karl A. P. Payne, Karl Fisher, Stephen A. Marshall, David Parker, Nicholas J. W. Rattray, Drupad K. Trivedi, Royston Goodacre, Stephen E. J. Rigby, Nigel S. Scrutton, Sam Hay, and David Leys. "UbiX Is a Flavin Prenyltransferase Required for Bacterial Ubiquinone Biosynthesis." Nature (2015): 502-06. Print. </p>
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<p>[2] Mckenna, Rebekah, Luis Moya, Matthew Mcdaniel, and David R. Nielsen. "Comparing in Situ Removal Strategies for Improving Styrene Bioproduction." Bioprocess Biosyst Eng Bioprocess and Biosystems Engineering (2014): 165-74. Print. </p>
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<p>[3] White, Mark D., Karl A. P. Payne, Karl Fisher, Stephen A. Marshall, David Parker, Nicholas J. W. Rattray, Drupad K. Trivedi, Royston Goodacre, Stephen E. J. Rigby, Nigel S. Scrutton, Sam Hay, and David Leys. "UbiX Is a Flavin Prenyltransferase Required for Bacterial Ubiquinone Biosynthesis." Nature (2015): 502-06. Print. </p>
 
<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 19:38, 26 September 2015

Styrene Synthesis Operon

Overview

This operon is a composite of the three enzymes involved in styrene synthesis in the following order: FDC (BBa_K1692002), UbiX (BBa_K1692007), and PAL (BBa_K1692004).The entire operon is controlled via an inducible T7 promoter. Each protein-coding sequence is preceded by a ribosome binding site and followed by a FLAG-tag peptide, enabling easy and efficient extraction

Styrene Synthesis Operon


Styrene synthesis pathway The enzymes of interest are phenylalanine ammonia lyase (PAL), ferulic acid decarboxylase (FDC), and a flavin prenyltransferase involved in ubiquinone biosynthesis called UbiX. PAL catalyzes the conversion of phenylalanine to trans-cinnamic acid, while FDC catalyzes the conversion of trans-cinnamic acid to styrene [2]. Recently, it has been discovered that a cofactor is required to activate FDC. This cofactor is a product of the reaction between dimethylallyl monophosphate (DMAP) and flavin mononucleotide (FMN), which is catalyzed by the enzyme UbiX [3].


Experiments and Results

The decision to place FDC first, PAL last, and UbiX in between was based on results from our mathematical model. Specifically, our model suggested that FDC exerts greatest control on the flux of biomaterials through the pathway; in other words, changes in FDC concentration result in the greatest changes in final styrene production. We combined this finding with the fact that earlier genes in an operon are expressed at higher levels [1]. Taken together, these results implied that we should place FDC first. Additionally, since UbiX’s cofactor product is essential for FDC activity, we decided to place UbiX immediately after FDC.



Once we determined the gene order for our FDC-UbiX-PAL operon, we needed to make it. To do this we used the New England Biolabs Gibson Assembly kit. We designed eight primers according to a primer design protocol (also from NEB) for FDC, UbiX, PAL and RFP plasmids. We then ran a PCR extension on all four of the resulting segments. We ran these four products on a gel and gel extracted the bands that appeared at the desired migration distances. We used this purified product for the Gibson assembly and transformed our resultant plasmid into NEB 5-alpha competent cells (NEB). We ran a colony PCR and found three colonies with the expected band length of over 4,000 base pairs. We grew up liquid cultures of these three colonies, miniprepped, and ran sequencing using five internal primers designed specifically for the combo plasmid. We found that two of the three colonies had the correct sequence!

We transformed the combo plasmid with FDC-UbiX-PAL operon (sequence confirmed) into T7 expressing competent cells (T7 Express, NEB) and performed a western blot using FITC-conjugated anti-flag antibody (Sigma) to confirm that all three genes were actually expressing the recombinant proteins with expected sizes.

Western blot analysis of in vivo expressed FDC-PAL-Ubix proteins

Confirming the clear over-expression of FDC over PAL protein from the FDC-UbiX-PAL operon in vivo, we are currently performing GC-MS analysis to see if the cells are fully capable of producing styrene at detectable and useful levels.

Reference

[1] Lim, H. N., Y. Lee, and R. Hussein. "Fundamental Relationship between Operon Organization and Gene Expression." Proceedings of the National Academy of Sciences (2011): 10626-0631. Print.

[2] Mckenna, Rebekah, Luis Moya, Matthew Mcdaniel, and David R. Nielsen. "Comparing in Situ Removal Strategies for Improving Styrene Bioproduction." Bioprocess Biosyst Eng Bioprocess and Biosystems Engineering (2014): 165-74. Print.

[3] White, Mark D., Karl A. P. Payne, Karl Fisher, Stephen A. Marshall, David Parker, Nicholas J. W. Rattray, Drupad K. Trivedi, Royston Goodacre, Stephen E. J. Rigby, Nigel S. Scrutton, Sam Hay, and David Leys. "UbiX Is a Flavin Prenyltransferase Required for Bacterial Ubiquinone Biosynthesis." Nature (2015): 502-06. Print.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 233
    Illegal BglII site found at 2154
    Illegal BglII site found at 3528
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 3746
  • 1000
    COMPATIBLE WITH RFC[1000]