Team:SDU-Denmark/Tour33

Design

The micro system

BioBrick BBa_K1088016 - pSB1A3-araC-HRT2-terminator. Device made to express the prenyltransferase, HRT2.

The most important step in the synthesis of natural rubber is the polymerization of isoprene catalyzed by HRT2. Our model showed that the reactions catalysed by Dxs and IspG are the rate limiting steps in the MEP pathway. Therefore it was decided that these would be the sites of our intervention in the pathway with the purpose of increasing the amount of IPP and DMAPP.

Our production system will be composed of two plasmids: one expressing HRT2 and one overexpressing Dxs and IspG.

Device expressing HRT2 - (pSB1K3-araC-Para-HRT2)
The expression of the device responds to arabinose under glucose scarce conditions; arabinose binds to AraC and relieves the transcription inhibition brought forth by this protein. It contains an arabinose promoter, a ribosomal binding site, the E.coli codon-optimized HRT2 gene of Hevea brasiliensis, and a terminator. The backbone is the pSB1K3 plasmid, which carries kanamycin resistance.

Biobrick BBa_K1088015 - pSB1C3-lacI-dxs-ispG. Device made to increase the IPP and DMAPP levels

Device expressing Dxs and IspG - pSB1C3-lacI-Plac-dxs-ispG
The device responds to allolactose and IPTG through LacI relieved inhibition. It contains a lactose promoter, a ribosomal binding site, the B. subtilis derived dxs gene, and the E. coli derived ispG gene. The backbone is the pSB1C3 plasmid, which carries chloramphenicol resistance.