Difference between revisions of "Team:Toronto/Composite Part"

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Revision as of 02:14, 19 September 2015

TodE and TodF Plasmid Design

Pseudomonas Putida F1 species degrade toluene with the help of tod operon. Toluene dioxygenase and toluene cis-dihydrodiol dehydroxygenase transcribed from transcribed from todC1C2BA and todD genes respectively facilitate break down of toluene to 3-methylcatechol. Gerben J. Zylstra et al successfully constructed the plasmid pTDG602 containing todC1C2BAD genes. To achieve complete degradation of toluene down to carbon dioxide and water, we focused on designing a plasmid containing todE and todF genes. After cotransformation of DH10B cells with pTDG602 and our plasmid, E.Coli theoretically should contain all the genes required to achieve complete degradation.

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In order to achieve the best protein production efficiency, we codon optimized our todE and todF sequences. We used Salis RBS calculator to optimize translation efficiency. We used a tunable promoter to determine optimum transcription for DH10B cells.

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We also used BioBricks in the registry to design plasmids with different efficiency by using RDP kit as shown below. In this process, we used promoter BBa_J23102 and RBS BBa_B0030.

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Note: Links to iGem registry BBa_J23102: http://parts.igem.org/Part:BBa_J23102 BBa_0030: http://parts.igem.org/Part:BBa_B0030