Magnetosome Production

1. We have PCR the flanking sequence of MFO, Recombination Template for mamAB Operon and Recombination Template for mamXY, mamGC and mms6 Operons (Figure 1).

Figure 1: The photo of 1% agarose gel electrophoresis. L: DNA ladder. Lane 1: PCR product of Recombination Template for mamAB Operon. Lane 2: PCR product of Recombination Template for mamXY, mamGC and mms6 Operons.

2. The PCR product of Recombination Template for mamAB Operon was then ligated into pSB1C3 backbone, forming K1648000, and ligated with promoter and double terminator in pSB1C3 backbone, forming BBa_K1648002. They were verified by double digestion (Figure 2) and sequencing.

Figure 2: Checking of recombinant plasmid using double digestion. L: DNA ladder. Lane 1-3: Recombination Template for mamAB Operon (K1648000) without digestion; with single digestion at XbaI site; with double digestion cut at XbaI and PstI site. Lane 4-6: Recombination Template for mamAB Operon with Promotor and Terminator (K1648002) without digestion; with single digestion at XbaI site; with double digestion cut at XbaI and PstI site.

3. The PCR product of Recombination Template for mamXY, mamGC and mms6 Operons was also ligated with promotor and double terminator in pSB1C3 backbone, forming K1648003. They were confirmed by double digestion (Figure 3) and sequencing.

Figure 3: Checking of recombinant plasmid using double digestion. L: DNA ladder. Lane 1-3: Recombination Template for mamXY, mamGC and mms6 Operons with Promoter and Terminator (K1648003) without digestion; with single digestion at XbaI site; with double digestion cut at XbaI and PstI site.

4. Expression of Recombination Template for mamAB Operon with Promoter and Terminator (K1648002). After introducing K1648002 into Azotobacter vinelandii by stable genome integration, every coding parts were successfully expressed. The expression of K1648002 was shown in SDS-PAGE (Figure 4).

Figure 4: SDS-PAGE showing expression of Recombination Template for mamAB Operon with Promotor and Terminator (BBa_K1648002) in Azotobacter vinelandii. L: Benchmark Protein ladder. Lane 1: Wild-type Azotobacter vinelandii. Lane 2: transformed Azotobacter vinelandii. Lane 2 shows 3 more bands compared to lane 1, in which the ~25 kDa band is the protein coded by chloramphenicol resistant gene in K1648002, the ~15 kDa and ~ 13kDa bands are the protein coded by Recombination Template for mamAB Operon.

5. Amplification of different operons from Magnetospirillum gryphiswaldense (MSR-1) by PCR (Figure 5). The PCR products were purified for homologous recombination later on.

Figure 5: The photo of 1% agarose gel electrophoresis showing PCR products of operons in Magnetosome Island (MAI). L: DNA ladder. Lane 1: mamHIEJKLMN. Lane 2: mamOPQRBSTU. Lane 3: mamPQRBSTU. Lane 4: mamYXZ ftsZ-like. Lane 5: mamGFDC. Lane 6: mamGFD. Lane 7: mms6 operon.

• Ongoing effort is the homologous recombination of K1648002 in transformed Azotobacter vinelandii.

1. We have made the Insertion Kit (Figure 6A) and amplified the GFP-nanobody (Figure 6B) by PCR.

Figure 6: The photo of 1% agarose gel electrophoresis showing PCR products. (A) L: DNA ladder. Lane 1: PCR products of linear Insertion Kit. (B) L: DNA ladder. Lane 2: GFP-nanobody.

2. The PCR product of GFP-nanobody was then ligated into pSB1C3 backbone, forming K1648005. Double digestion (Figure 7) and sequencing verified it.

Figure 7: Checking of recombinant plasmid using double digestion. L: DNA ladder. Lane 1-3: GFP- nanobody (K1648005) without digestion; with single digestion at XbaI site; with double digestion cut at XbaI and PstI site.

3. J04450 was inserted into Insertion Kit, forming Insertion Kit for Fusing Protein of Interest to Magnetosome Membrane (K1648004) fulfilled the biobrick standard, while GFP-nanobody (K1648006) was also added into Insertion Kit respectively. Double digestion (Figure 8) shows the expected result.

Figure 8. Checking of recombinant Insertion Kit for Fusing Protein of Interest to Magnetosome Membrane (K1648004) using double digestion. (A) L: DNA ladder. Lane 1-3: Insertion Kit for Fusing Protein of Interest to Magnetosome Membrane (K1648004) without digestion; with single digestion at XbaI site; with double digestion cut at XbaI and PstI site. (B) L: DNA ladder. Lane 1-3: Insertion kit with GFP-nanobody (K1648006) without digestion; with single digestion at XbaI site; with double digestion cut at XbaI and PstI site.

• Current progress is the characterization of mamC-GFP nanobody fused protein.