Tardigrades are microscopic animals that can be found worldwide in both aquatic and terrestrial habitats. They gather attention recently with their unique adaptation to a range of extreme environments. Most important for us is the fact that tardigrades can avoid freezing by supercooling to below -20 °C(3). It has been shown that synthesis of bioprotectants in the form of selective proteins plays a role in their survivability. Such putative protective proteins are: Cytosolic abundant heat soluble protein 1 (CAHS1)(1,2) , Secretary abundant heat soluble protein 2 (SAHS2) (1,2), Mitochondrial abundant heat soluble protein (MAHS) (1,2), and Late embryogenesis abundant protein, mitochondrial (RvLEAM) (1,2).

Our project is an attempt to mimick the tardigrades unique survivability in different bacteria species via heterologous expression of protective proteins. We took the four previously mentioned proteins, after that we selected their longest isoforms and optimized these sequences for expression in E.coli. These constructs were ordered as gBlocks from IDT. Using iGEM parts delivered to us, we amplified an inducible promoter (tac-lacO) cassette and cloned it in front of our synthesized sequences using overlap extension PCR.

We took the generated constructs and electroporated them into Rosetta gammi strain (optimized for protein expression). Expression levels were monitored with SDS-PAGE. The cryoprotective potential of our devise was tested in freeze and thaw experiments followed by cell viability quantification.

Our further plans involve testing of our synthetic construct in Lactobacillus delbrueckii subsp. bulgaricus, which is endemic for Bulgaria and a crucial producer in our food industry.

REFERENCES:

1. Ayami Yamaguchi, Sae Tanaka, Shiho Yamaguchi, Hirokazu Kuwahara, et al. (August 28, 2012); Two Novel Heat-Soluble Protein Families Abundantly Expressed in an Anhydrobiotic Tardigrade; PLoS ONE 7(8): e44209. doi:10.1371/journal.pone.0044209
2. Sae Tanaka, Junko Tanaka, Yoshihiro Miwa, Daiki D. Horikawa ,Toshiaki Katayama, et al. (February12,2015) Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells; PLoS ONE; doi:10.1371/journal.pone.0118272
3. Acta Physiol (Oxf). 2011 Jul;202(3):409-20. doi: 10.1111/j.1748-1716.2011.02252.x. Epub 2011 Mar 22.