Difference between revisions of "Team:Uppsala/Design"
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<li>UDP-glucuronosyltransferase (UGT) catalyzes the glucuronidation reaction of forming crocin from crocetin <a href="#ref3">(3)</a>.</li></ol> | <li>UDP-glucuronosyltransferase (UGT) catalyzes the glucuronidation reaction of forming crocin from crocetin <a href="#ref3">(3)</a>.</li></ol> | ||
<br> | <br> | ||
| − | Next we researched for the most appropriate enzymes under the above enzyme classes to execute the successful conversion of all the intermediates to crocin. We found CaCCD2 <a href="#ref4">(4)</a>, CsADH2946 <a href="#ref3">(3)</a>and UGTCs2 <a href="#ref5">(5)</a> to be the most promising for our project.<br> | + | Next we researched for the most appropriate enzymes under the above enzyme classes to execute the successful conversion of all the intermediates to crocin. We found CaCCD2 <a href="#ref4">(4)</a>, CsADH2946 <a href="#ref3">(3)</a> and UGTCs2 <a href="#ref5">(5)</a> to be the most promising for our project.<br> |
<br> | <br> | ||
The genes of the three individual enzymes were identified and synthesized as a gBlocks by IDT. Prior to synthesis, the genes were also codon optimized for <i>E. coli</i>, to get the desired overexpression of our enzymes. An N-terminal His-tag was added to aid downstream purification process. This was done after drawing conclusions from the <a href="https://2017.igem.org/Team:Uppsala/Model">modeling results</a> that predicted the N-terminal to be outside of the enzymes and far from the active sites after folding. The promoter used was an inducible promoter <a href="http://parts.igem.org/Part:BBa_J04500">BBa_J04500</a> from the iGEM kit. The enzymes were created into BioBricks using plasmid pSB1C3-J04500 with IPTG-inducible expression. The plasmid was linearised with <a href="https://2017.igem.org/Team:Uppsala/Experiments">Phusion PCR</a> and the constructs were inserted into the iGEM plasmid using <a href="https://2017.igem.org/Team:Uppsala/Experiments">Gibson assembly</a>. We <a href="https://2017.igem.org/Team:Uppsala/Experiments">transformed</a> the plasmid into <i>E. coli</i> (TOP10 competent cells), screened the colonies using <a href="https://2017.igem.org/Team:Uppsala/Experiments">colony PCR</a> and run <a href="https://2017.igem.org/Team:Uppsala/Experiments">gel electrophoresis</a> to validate that the insert had been successfully assembled into the plasmid. We also created BioBricks with a part for RFP expression. | The genes of the three individual enzymes were identified and synthesized as a gBlocks by IDT. Prior to synthesis, the genes were also codon optimized for <i>E. coli</i>, to get the desired overexpression of our enzymes. An N-terminal His-tag was added to aid downstream purification process. This was done after drawing conclusions from the <a href="https://2017.igem.org/Team:Uppsala/Model">modeling results</a> that predicted the N-terminal to be outside of the enzymes and far from the active sites after folding. The promoter used was an inducible promoter <a href="http://parts.igem.org/Part:BBa_J04500">BBa_J04500</a> from the iGEM kit. The enzymes were created into BioBricks using plasmid pSB1C3-J04500 with IPTG-inducible expression. The plasmid was linearised with <a href="https://2017.igem.org/Team:Uppsala/Experiments">Phusion PCR</a> and the constructs were inserted into the iGEM plasmid using <a href="https://2017.igem.org/Team:Uppsala/Experiments">Gibson assembly</a>. We <a href="https://2017.igem.org/Team:Uppsala/Experiments">transformed</a> the plasmid into <i>E. coli</i> (TOP10 competent cells), screened the colonies using <a href="https://2017.igem.org/Team:Uppsala/Experiments">colony PCR</a> and run <a href="https://2017.igem.org/Team:Uppsala/Experiments">gel electrophoresis</a> to validate that the insert had been successfully assembled into the plasmid. We also created BioBricks with a part for RFP expression. | ||
Revision as of 16:36, 1 November 2017
The saffron biosynthetic pathway is an extension of the β-carotene pathway, with zeaxanthin being a key intermediate (1). The pathway from farnesyl pyrophospate (FPP) to zeaxanthin has been BioBricked before, but we wanted to extend the β-carotene pathway to continue from zeaxanthin to crocin.
Plan integrating FPP to Zeaxanthin in Chromosome
A BioBrick from iGEM Slovenia 2010 coding for zeaxanthin includes a set of five genes, thus making it a very large operon. As we wanted to extend the strain by adding three more genes we realised this would make the strain very unstable. To solve this our team created a zeaxanthin producing strain with chromosome integration of the genes using lambda red. Read about the process and results here!
Plan going from Zeaxanthin to Crocin
To extend the pathway for the conversion of zeaxanthin to crocin, we needed to add the three step pathway, catalyzed by three different enzyme classes:
Next we researched for the most appropriate enzymes under the above enzyme classes to execute the successful conversion of all the intermediates to crocin. We found CaCCD2 (4), CsADH2946 (3) and UGTCs2 (5) to be the most promising for our project.
The genes of the three individual enzymes were identified and synthesized as a gBlocks by IDT. Prior to synthesis, the genes were also codon optimized for E. coli, to get the desired overexpression of our enzymes. An N-terminal His-tag was added to aid downstream purification process. This was done after drawing conclusions from the modeling results that predicted the N-terminal to be outside of the enzymes and far from the active sites after folding. The promoter used was an inducible promoter BBa_J04500 from the iGEM kit. The enzymes were created into BioBricks using plasmid pSB1C3-J04500 with IPTG-inducible expression. The plasmid was linearised with Phusion PCR and the constructs were inserted into the iGEM plasmid using Gibson assembly. We transformed the plasmid into E. coli (TOP10 competent cells), screened the colonies using colony PCR and run gel electrophoresis to validate that the insert had been successfully assembled into the plasmid. We also created BioBricks with a part for RFP expression.
Plan integrating FPP to Zeaxanthin in Chromosome
A BioBrick from iGEM Slovenia 2010 coding for zeaxanthin includes a set of five genes, thus making it a very large operon. As we wanted to extend the strain by adding three more genes we realised this would make the strain very unstable. To solve this our team created a zeaxanthin producing strain with chromosome integration of the genes using lambda red. Read about the process and results here!
Plan going from Zeaxanthin to Crocin
To extend the pathway for the conversion of zeaxanthin to crocin, we needed to add the three step pathway, catalyzed by three different enzyme classes:
- Carotenoid cleavage dioxygenases (CCD) are responsible for the symmetric cleavage of zeaxanthin at the 7,8/7′,8′ positions to form crocetin dialdehyde from zeaxanthin (2).
- Aldehyde dehydrogenases (ALDH) converts the 20 carbon cleavage product, crocetin dialdehyde to crocetin (3).
- UDP-glucuronosyltransferase (UGT) catalyzes the glucuronidation reaction of forming crocin from crocetin (3).
Next we researched for the most appropriate enzymes under the above enzyme classes to execute the successful conversion of all the intermediates to crocin. We found CaCCD2 (4), CsADH2946 (3) and UGTCs2 (5) to be the most promising for our project.
The genes of the three individual enzymes were identified and synthesized as a gBlocks by IDT. Prior to synthesis, the genes were also codon optimized for E. coli, to get the desired overexpression of our enzymes. An N-terminal His-tag was added to aid downstream purification process. This was done after drawing conclusions from the modeling results that predicted the N-terminal to be outside of the enzymes and far from the active sites after folding. The promoter used was an inducible promoter BBa_J04500 from the iGEM kit. The enzymes were created into BioBricks using plasmid pSB1C3-J04500 with IPTG-inducible expression. The plasmid was linearised with Phusion PCR and the constructs were inserted into the iGEM plasmid using Gibson assembly. We transformed the plasmid into E. coli (TOP10 competent cells), screened the colonies using colony PCR and run gel electrophoresis to validate that the insert had been successfully assembled into the plasmid. We also created BioBricks with a part for RFP expression.
References
- Ángela, L. G., and Ahrazem, R. O. 2010. Understanding Carotenoid Metabolism in Saffron Stigmas : Unravelling Aroma and Colour Formation. Functional Plant Science and Biotechnology 4, 56–63.
- Frusciante S, Diretto G, Bruno M, Ferrante P, Pietrella M, Prado-Cabrero A, et al. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis. Proceedings of the National Academy of Sciences. 2014 Aug 19;111(33):12246–51.
- Gómez-Gómez L, Parra-Vega V, Rivas-Sendra A, Seguí-Simarro JM, Molina RV, Pallotti C, et al. Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma. Int J Mol Sci [Internet]. 2017 Jan 1 [cited 2017 Oct 29];18(1).
- Ahrazem O, Rubio-Moraga A, Berman J, Capell T, Christou P, Zhu C, et al. The carotenoid cleavage dioxygenase CCD2 catalysing the synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme. New Phytol. 2016 Jan 1;209(2):650–63
- Moraga AR, Nohales PF, Pérez JAF, Gómez-Gómez L. Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas. Planta. 2004 Oct;219(6):955–66.
