Team:Hong Kong-CUHK/Results

Result

Highlights

• We 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 was made as a biobrick (K1648006). Also, GFP-nanobody has been added into Insertion Kit for characterization. Other teams who are working with magnetosome could employ the present Insertion Kit to express various proteins on magnetosome!

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.




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 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.


Characterization of Lead-binding Peptide (LBP) Efficiency


Lead-binding peptide (LBP) TNTLSNN was designed to bind with Ni-ATA by adding His-tag (HHHHHH). We generated four set of peptides:

1. 1×LBP-6xHis TNTLSNNHHHHHH
2. 2×LBP-6xHis TNTLSNNTNTLSNNHHHHHH
3. 2×LBP-linker-6xHis TNTLSNNGGGTNTLSNNHHHHHH
4. 1×LBP-linker-6xHis TNTLSNNGGGHHHHHH


to investigate whether (1) number of LBP; (2) linker between lead binding site and His-tag site affect the lead binding efficiency. Final concentration of 1 mM lead nitrate solution mixed with 1 mg of each peptide in total 1 ml was incubated for 1 h at RT. Negative control: 1. peptide without lead nitrate solution; 2. lead nitrate solution only; 3. buffer only was setup to assess the lead binding effect of the peptides. Ni-ATA resin (200 μl; cOmpleteTM His-Tag purification resin) was used to capture the peptides. The resin was washed three times with Ni-ATA buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0), and eluted with 1 ml Ni-ATA buffer plus 100 mM imidazole. Elution (150 μl) was mixed with 450 μl concentrated nitric acid, incubated for 24 h at 60 oC, and loaded into Atomic Absorption Spectrometer (AAS). Lead nitrate solution standards were prepared to calculate the lead concentration in samples.


Compared to the negative control 1. peptides only and 3. buffer only, higher Pb concentration of elution was found in 2. Pb only control. It indicated there is either non-specific binding of Pb on the Ni-ATA resin or incomplete washing. Compared peptide+Pb samples with 2. Pb only control, 1×LBP-6xHis with or without linker showed higher concentration of Pb binding than that with 2×LBP, suggesting 2×LBP may hinder the 3D-configuration for Pb binding.