Team:Exeter/Results/showcase

Showcasing Polysaccharide Production - Becca Philp

The unique and variable properties of polysaccharides in nature led us to attempt production of a variety of polysaccharides from different natural sources into our E. coli. These polysaccharides have variable structures and important applications in diverse business sectors. The Hyaluronan synthase and cyclodextrin glycosyltransferase enzymes were successfully cloned and sequenced inside pSB1C3 plasmids and submitted to the registry for future teams to realise their potential products (BBa_K764022 and BBa_K764026 respectively).

Using 3A assembly we then proceeded to attempt production of the gene constructs for expression in our E coli. Each of the showcase enzymes were successfully linked to terminators on their own. We attempted the linking of the enzymes to promoter-RBS sequence but due to time constraints were unable to sequence the parts and prove our constructs had been produced.

Newcastle iGEM 2010’s biobrick BBa_K322921 was adapted by adding to the signal peptide OmpA and promoter-RBS sequence. These sequences were confirmed in pSB1C3 plasmids and submitted to the registry as:

• BBa_K764033 Tetr promoter_rbs_ompA_SacB-Term
• BBa_K764034 Pbad Weak promoter_rbs_ompA- SacB-Term
• BBa_K764026 Cyclodextrin glycosyltransferase + double terminator
• BBa_K764025 Hyaluronan synthase + double terminator
• BBa_K764024 SacB + double terminator

The linking of Levansucrase, the adaption of Newcastle iGEM 2010’s gene for SacB, to a signal peptide ompA, was to allow the enzyme to be exported from the cell. This was for two reasons, firstly to demonstrate that our polysaccharides could be produced both intracellular and extracellularly, but also because levansucrose is toxic to E. coli so we wanted to allow its production by E.coli without damaging the cells. We were however unable to add the construct to an expression vector to demonstrate this. The submission of OmpA, is the first signal peptide to the registry.

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