The construction of the MFO

Our first construct is called the MFO, which stands for the magnetosome forming operon. Different operons that are used to produce and regulate the magnetosome formation[1]. Among them, it is the mamAB operon that is the crucial operon that is needed for the production of magnetosome. Other operons like mamGFDC, mamXY, mms6 et cetera are more frequently used for regulating the size and shape and the biominerlization that facilitate the formation of magnetosome[2].
Therefore, we would only insert the mamAB operon from Magnetospirillum gryphiswaldense (MSG) to bacteria Azotobacter Vinelandii, hoping to use minimal number of genes to produce functional magnetosome. Future insight is given to make the magnetosome formation progress easier.
Considering the rather large size of mamAB operon, which is 16.4 KB, a new method was employed to help its transfer into the Azotobacter vinelandii. The overview of mamAB construct cloning strategy is shown in Figure 1.

Figure 3: Cloning strategy for mamAB operon

The insertion kit:

This construct consists of a mamC gene, a gene coding for a trans-membrane protein [3] on the magnetosome membrane. Unlike usual recombinant methods in which insert is put between the multiple restriction sites, we are putting our mamC gene in front of them. Thus it enables the attachment of any protein desired to the magnetosome membrane, through insertion into the multiple restriction sites fusing with mamC. (For your interest, this is done by removing the stop codon of the mamC gene and the start codon of the desired protein, for example an antibody, making it a mamC fused protein).

Normal method of inserting the gene of interest by restriction digestion and ligation is not possible in here. Instead, the whole plasmid has to be synthesized by ourselves using PCR method. The overview of cloing strategy is summarized in Figure 2. Four fragments would be produced firstly: B0015 – the double terminator; backbone with kanamycin; J13002 (which consists of the constitutive promoter R0040 and the RBS); and mamC.
Through the over-lapping PCR of these four fragments, one linear fragment would be produced. And we have designed PstI sites at both the start and the end of the fragment. Consequently, by cutting the PstI site and ligate it, a circular plasmid will be produced.

Figure 4: Pstl ligation

[1]. LOHßE, Anna, et al. Genetic dissection of the mamAB and mms6 operons reveals a gene set essential for magnetosome biogenesis in Magnetospirillum gryphiswaldense. Journal of bacteriology, 2014, 196.14: 2658-2669.

[2]. CHEN, Zhucheng; YANG, Haijuan; PAVLETICH, Nikola P. Mechanism of homologous recombination from the RecA–ssDNA/dsDNA structures. Nature, 2008, 453.7194: 489-494.

[3]. XU, Jun, et al. Surface expression of protein A on magnetosomes and capture of pathogenic bacteria by magnetosome/antibody complexes. Frontiers in microbiology, 2014, 5.