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Team:Kyoto/Project/Magnetosome Formation
From 2014.igem.org
MAGNETOSOME FORMATION
Introduction
We, iGEMer normally use Escherichia coli (E. coli) for introducing transgene. We introduce valuable genes into E. coli in order to live more comfortable life using E. coli which has special ability. However, we cannot easily spread special E. coli which we make, because they happen to effect environment or ecosystem. We have to carefully think about these things. We, iGEM Kyoto team members, are hit by fantastic idea. If we make E. coli which can be collected by magneto, we can spread special E. coli without considering environment or ecosystem. We read a lot of papers to make E. coli which can be collected by magneto and found magnetotactic bacteria which have magneto in their body. Then we have decided to make effort to produce E. coli which have magnet (We named them Magneto coli) using genes of magnetotactic bacteria.
Magnetotactic bacteria have magneto in a small vesicle. This structure is called magnetosome. Size of magnetosome is about 50 nm – 100 nm and magnetite (Fe3O4) and phospholipid, which covers magnetite, consist of magnetosome. So some scientists think that magnetosome is primitive organelle. There are variety types of magnetotactic bacteria—AMB-1, MSR-1, MS-1 and so on. They all are microaerophile (organism that thrives in an environment low in oxygen) and they use magnetosome for swimming to place they can live. How they use magnetosome? Thanks to magnetosome, they can sense geomagnetism and a line of magnetic force and they swim to the North Pole when they are in the northern hemisphere and swim to the South Pole when they are in the southern hemisphere. And they use magnetosome in the other way. Thanks to magnetosome, they can receive geomagnetism and a linear magnetic force and swim deeper sea where oxygen (O2) level is low. After all we can say that magnetosome is compass which guides magnetotactic bacteria to place where they can live.
It is well known that gene clusters which have something to do with biosynthesis of magnetosome congregate in one area called magnetosome island (MAI). There are four important operons (mamAB operon, mamGFDC operon, mamXY operon, and mms6 operon) in MAI. There are three main reasons why genes in these four operons are thought that they are something to do with biosynthesis of magnetosome. First there are variety types of magnetotactic bacteria. However, MAI is highly conserved between these magnetotactic bacteria. Second magnetotactic bacteria which have mutation in genes on MAI cannot make magnetosome, have smaller or fewer magnetosomes than wild type. For example, one of magnetotactic bacteria – AMB-1 which are knocked out part of mamAB operon cannot make magnetosome. Third photosynthetic bacteria R. rubrum (of course wild type don’t have magneto in their body). get ability to produce magnetosome when they are introduced four important operons in MAI and one gene (feoB1).
Biosynthesis of magnetosome is roughly divided into five stages. First MamB, MamL, MamQ proteins induce vesicle biogenesis. Second two proteins sort in magnetosome membrane. Third five proteins induce uptake of iron and magnetite crystal nucleation. Fourth a lot of proteins in MAI induce magnetosome maturation. Fifth three proteins induce magnetosome assembly and positioning.
We 2014 iGEM Kyoto members have decided to choice AMB-1 from variety types of magnetotactic bacteria. This is because AMB-1 is stronger than other magnetotactic bacteria and we think that the stronger magnetosome is in aerobic condition, the more easily we produce Magneto coli. There are other reasons. We happened to notice that 2013 OUC-China conducted project which like to our project and they used AMB-1. We have appreciated this project. We want to make magneto in E. coli and it is necessary that magnet (Fe3O4) is covered by vesicle (OUC-China made effort to make this vesicle.). So, we have thought that if we approve OUC-China’s project, we can make magnetosome in E. coli. We would like to everyone, who read our wiki, our improvement plan. First we tried to identify genes which are something to do with biogenesis of magnetosome. OUC-China introduced five genes, while we introduced three genes (mamB, mamQ and mamL). Second we used electron microscopy (We used TEM in Kyoto University).
Experiments & Results
In order to reproduce the vesicle biogenesis in side E. coli, we first cloned the three genes—mamL, mamQ, and mamB from magnetotactic bacteria strain AMB-1. And then we modified each of these three genes by adding upstream the 5’ end an efficient RBS B0034 and replacing stop codon with a Histidine tag (six continuous Histidine codons plus a stop codon). Using each one of these modified genes, we then constructed three plasmids with promoter J23100 and backbone plasmid pSB1C3 (with terminator). The three plasmids—pL, pQ and pB, for short, transferred separately into E. coli, were constructed to make sure genes mamL, mamQ, and mamB is potential to be expressed under the E. coli metabolism. Protein MamL, MamQ, and MamB are successfully detected by Histidine tag antibody and the results are showed by Western Blotting.
Under the expression evidence provided by Western Blotting, we started the construction of the plasmid pLQB. Modified mamQ with RBS was cut from pQ and then inserted into pL to make pLQ. Same way, Modified mamB and RBS was cut from pB and inserted into pLQ to make pLQB. For every plasmid we sequenced them to make sure no mutation and insertion were introduced during construction processes (especially PCR).
Eventually we completed the final plasmid pLQB and transferred it into E. coli. After E. coli colonies grow on the plates, we examined each colony by colony PCR hoping to fide one containing pLQB. It was really desperate when we examine 48 colonies finding nothing. Yet hard work pays off after another round of colony PCR, we finally detected pLQB in a relatively small colony. The final plasmid pLQB underwent Histidine tag antibody Western Blotting and Sanger sequencing. According to result, three proteins are expressed concurrently and DNA sequence was still untouched.
Finally we could observe our transgenic E. coli under TEM.
Discussion
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Conclusion
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Future Work
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Reference
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