'Part Ⅱ: Fusion Proteins for Transportation into BMC'

It is reported that a short N-terminal peptide is necessary and sufficient for packing enzymes into the lumen of the BMC. Fusion of the 14, 18 or 64 N-terminal amino acids from PduP to GFP or RFP resulted in their encapsulation within BMCs. Each fusion protein (PduP[1-14]- GFP, PduP[1-18]- GFP, PduP[1-64]- GFP) was produced by the new standard. Our new standard, as it is stated above, consists of three restriction enzyme cut sites --- SacⅠ(CCTCG)，EarⅠ(CTCTTC) and SapⅠ(GCTCTTC). The details of enzyme digestion and ligation can be seen in Fig 2a. We used GFP plasmid as a vector and P_L plasmid as an insert. Through the 4h enzyme digestion of the new standard, we got two expected segments. However, the base pairs of the two segments are not complementary. Thus, when came to the ligation, a linker (10*GS) was used. When 14/18/64 amino acids from the N terminus of PduP were fused to GFP, the fusion protein was readily detected by Western blotting.