Results

The final goal of our project was to develop a system capable to improve a microorganism’s resistivity to extreme osmolarity and/or temperature conditions. For that purpose we needed to identify putative proteins capable of providing such properties to living organisms and also to create a straightforward manner for their expression in the targeted microorganisms.

After a thorough literature research we have identified the tardigrades as most prominent species known for their capacity to survive freezing and desiccation. Eventually we selected the following proteins for expression in microorganisms:

• Cytosolic abundant heat soluble protein 1 (CAHS1)
• Secretary abundant heat soluble protein 2 (SAHS2)
• Mitochondrial abundant heat soluble protein (MAHS)
• Late embryogenesis abundant protein, mitochondrial (RvLEAM)

The protein sequences were codon-optimized and were synthesized with appropriate flanking restriction sites (BioBrick’s prefix and suffix - Fig. 1).

Fig. 1 Schematic view of the used gBlock fragments. CDS are the optimized coding sequences of our four proteins.

Those synthetic genes were consequently used for the construction of expression cassettes, which were controlled by a tac promotor (repressible by lacI) and inserted in pSB1A3 vectors (Fig. 2).

Fig. 2 Schematic view of the used expression cassettes. Inducible tac-lacO promoter drives the transcription of our sequences. Each cassette also has the LacI regulator oriented in the opposite direction. The pSB1A3 vector delivers transcriptional terminators at both sides of each cassette.

The plasmids thusly obtained were electroporated in Top10 E. coli strains for plasmid multiplication and maintenance.

Next step was to introduce the expression plasmids in Rosetta gammi E. coli strains for improved protein expression levels. The expression levels of the putative protective proteins were measured through SDS-PAGE. Finally, our engineered bacteria were tested for improved viability after freeze-thaw cycles.