DMS SYNTHESIS

Introduction

Everyone knows Cloud is just a visible mass of water or ice particle and we can’t stand on cloud. However, it is not just a mass of water or ice particle. Water particles are insufficient to make up cloud, Cloud Condensation Nuclei (CCN) is also necessary to make cloud. CCN is a collective term for tiny particles upon which water vapor condenses, and most CCN are aerosol. When water vapors condenses in the air, they concentrate on CCN and make up cloud.

In those CCN aerosol, sulfates are included.　Sulfate aerosols are included in smoke emitted by factories. However, some marine organisms are also producing a precursor of sulfates, dimethyl sulfide (DMS).DMS biosynthesis is not done by a single species. It is a combination work of some species, for example, corals or diatoms produce a precursor of DMS, dimethylsulfoniopropionate (DMSP), and marine bacteria synthesize DMS from DMSP. In the natural world, DMS becomes CCN in this way; DMS is biosynthesized in the sea and do up sky for its volatility. Then it is exposed by ultraviolet rays and becomes sulfates through multistep reaction. Finally, water vapors condenses around it.

Then, we focused on DMS and thought producing DMS can contribute cloud forming. We considered E. coli can imitate DMS biosynthesis and produce DMS. To make E. coli being able to produce DMS, we investigated papers and found organisms which have those biosynthesis pathway.

We owe Barbara R. Lyon et al.’s work in 2011[1] a lot to decide what genes we introduce in E. coli to construct Met to DMSP biosynthesis pathway. In the work, Fragilariopsis cylindrus, a model sea-ice diatom, was selected to investigate. Their osmotic pressure is controlled by DMSP density, so comparison of protein responses between F. cylindrus grown in high-salinity and general-salinity was done in the paper. Protein responses is confirmed by two-dimensional electrophoresis, and mass spectrometry was done to spots confirmed the increase of express. From this, 36 candidates were picked up. Then, they researched databases and classification of protein, and 18 genes are survived as candidates. In those genes, 13 genes were eliminated as working in irrelevant biosynthesis pathway. Finally, 5 candidate genes are presented and assigned to each step.

Owing to this work, we decided to introduce these 5 genes from F. cylindrus to E. coli. We also introduced dddD gene from Ruegeria pomeroyi. DMSP to DMS pathway enzyme is coded by dddD gene[2]. In Jonathan D. Todd et al.’s work in 2007[3], it is confirmed that dddD gene from Marinomonas sp. MWYL1 is functional in E.coli. From its highly homology, we decided to use R. pomeroyi's gene.

Summarizing our project, we introduce 6 genes relating to the Met-DMSP pathway and DMSP-DMS pathway and make E.coli produce DMS. However, we don’t know how it works well by introducing 6 genes all at once. Then, we decide to confirm whether each gene’s function at first. To confirm functions, we used High Performance Liquid Chromatography (HPLC) and compared HPLC data of pure products and homogenate of each precursor and transformant and mixture of each precursor and purified protein. We used DMS detecting tube to confirm function of dddD gene. Because we thought too much noises may appear in HPLC data of the final reaction.

Experiments & Results

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Discussion

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Conclusion

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Future Work

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Reference

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Team:Kyoto/Project/DMS Synthesis

From 2014.igem.org

DMS SYNTHESIS

Introduction

Experiments & Results

Discussion

Conclusion

Future Work

Reference