From 2007.igem.org

We propose to link the riboflavin operon of Bacillus subtilis to plasmid puc18-ADH1 p+t(figure1). ADH1p(560bp) is a strong promoter of Yeast and ADH1t is the terminater corresponding to this promoter. The initial carrier of puc18-ADH1 p+t is PUC18. The construction of this plamid is:

1. Digest ADH1p with XbaI and BAMHI and link it to the relevant site of PUB18;
2. Digest ADH1t with KpnI and SacI and link it to the relevant site of PUC18.

Our main work is :

1. Design the primer and amplify the riboflavin operon of bacillus subtilis. The restriction site of up- and down-primer are SmaI(Cfr9I) and KpnI, respectively.
2. Digest the PCR product with SmaI(Cfr9I) and KpnI, and then link it to plasmid puc18-ADH1 p+t.
3. Digest the linked ADH1p+rib operon+ADH1t(5kb) fragment with XbaI and EcoRI, and link it to plasmid Yeplac195(figure2)
4. Trandfer the constructed Yeplac195 into Yeast
5. Test the expression of riboflavin operon of Bacillus subtilis in Yeast and the yield of accumulated riboflavin.