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

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           <p>We decided to use four different promoters to drive expression of our pili proteins. The T7 promoter (BBa_I712074) was chosen as it is a strong, well characterised promoter but as this could only be used in BL21(DE3) it was only used for initial expression studies. Two inducible promoters were chosen to allow control over expression in non pili-producing strains of <i>E. coli</i>: the rhamnose inducible promoter P_Rha (BBa_K902065) and the arabinose inducible promoter P_ara (BBa_I13453). Finally the constitutive promoter P_J23100 (BBa_J23100) was chosen.</p>
 
           <p>We decided to use four different promoters to drive expression of our pili proteins. The T7 promoter (BBa_I712074) was chosen as it is a strong, well characterised promoter but as this could only be used in BL21(DE3) it was only used for initial expression studies. Two inducible promoters were chosen to allow control over expression in non pili-producing strains of <i>E. coli</i>: the rhamnose inducible promoter P_Rha (BBa_K902065) and the arabinose inducible promoter P_ara (BBa_I13453). Finally the constitutive promoter P_J23100 (BBa_J23100) was chosen.</p>

Revision as of 16:00, 30 October 2017

Composite Parts

We decided to use four different promoters to drive expression of our pili proteins. The T7 promoter (BBa_I712074) was chosen as it is a strong, well characterised promoter but as this could only be used in BL21(DE3) it was only used for initial expression studies. Two inducible promoters were chosen to allow control over expression in non pili-producing strains of E. coli: the rhamnose inducible promoter P_Rha (BBa_K902065) and the arabinose inducible promoter P_ara (BBa_I13453). Finally the constitutive promoter P_J23100 (BBa_J23100) was chosen.

We chose the well characterised strong RBS BBa_B0034 and the double terminator BBa_B0015.

All parts were synthesised as gBlocks by IDT and composite parts were built using a one-pot cloning method *protocol* modified from Weber et al 2011. All FimH constructs were inserted into pSB1A3 and the film operon constructs were inserted into pX1’6’0’0 (lab built plasmid). The parts listed below have been submitted to iGEM in pSB1C3.

FimH sfGFP

Name Description Base Pairs
BBa_K2324007 P_Rha_FimH_22_sfGFP 2020
BBa_K2324006 P_Rha_FimH_225_sfGFP 2020
BBa_K2324008 P_Rha_FimH_258_sfGFP 2020
BBa_K2324011 P_T7_FimH_225sfGFP 1798

FimH Fusion Proteins

Name Description Base Pairs
BBa_K2324002 P_Rha_FimH_22_His 1102
BBa_K2324001 P_Rha_FimH_22_SynMT 1474
BBa_K2324003 P_Rha_FimH_22_MouseMT 1489

Fim Operon

Name Description Base Pairs
BBa_K2324012 P_J23100_Fim_Operon 5944
BBa_K1850013 P_Ara_Fim_Operon 5874
Reference

Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marrillonnet, S. (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS One 6, e16765