Team:Hong Kong-CUHK/Results

Result:

Highlights

• We have made the templates with flanking sequences of Magnetosome Forming Operons (MFO) for homologous recombination. The templates were successfully integrated into Azotobacter vinelandii genome and successfully expressed.

• The insertion kit has been made as a biobrick (BBa_K1648006). Also, GFP-nanobody gene has been added to Insertion Kit for characterization of it. Other teams who are working with magnetosome could employ the present Insertion Kit to express various proteins on magnetosome!

Magnetosome

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


2. The PCR product of Recombination Template for mamAB Operon was then ligated into pSB1C3 backbone, forming BBa_K1648000, and ligated with promotor 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 (BBa_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 (BBa_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 mms Operons was also ligated with promotor and double terminator in pSB1C3 backbone, forming BBa_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 mms Operons with Promotor and Terminator (BBa_K1648003) without digestion, with single digestion at XbaI site, with double digestion cut at XbaI and PstI site.


4. Expression test of Recombination Template for mamAB Operon with Promotor and Terminator (BBa_K1648002). After introducing BBa_K1648002 into Azotobacter vinelandii by stable genomic integration, every coding parts were successfully expressed. The expression of BBa_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: 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 BBa_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: mamYXZftsZ-like. Lane 5: mamGFDC. Lane 6: mamGFD. Lane 7: mms6.


• Ongoing effort is the homologous recombination of BBa_K1648002 transformed Azotobacter vinelandii.




Insertion Kit


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 BBa_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 (BBa_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 (BBa_K1648004) fulfilled the biobrick standard, while GFP-nanobody (BBa_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 (BBa_K1648004) using double digestion. (A) L: DNA ladder. Lane 1-3: Insertion Kit for Fusing Protein of Interest to Magnetosome Membrane (BBa_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 (BBa_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.