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Freeze-Drying Device

Bactoblood needs the ability to be stored for long periods in a freeze-dried form so that it can be stockpiled and easily transported. We therefore sought a genetic device that could improve the Bactoblood's structural integrity during dessication. We designed two distinct desiccation devices: one that utilizes the disaccharide trehalose and one that involves the small molecule hydroxyectoine. Molecules have been demonstrated to provide protection against damage due to dessication and freeze drying. The figure below shows our actual lyophilized Bactoblood. The white chunks are crystallized sucrose, which was used as a protectant.

Trehalose

The genes in the trehalose biosynthesis pathway are pulled right out of the E.coli genome and consist of otsA and otsB. By overexpressing these genes after bactoblood has reached saturation, the can protect itself from damage during freeze drying without having to add exogenous protectants. OtsB encodes for a 29.1-kDa trehalose-6-phosphate phosphatase and otsA encodes for a 53.6-kDa trehalose-6-phosphate synthase. Together they make up a trehalose-6-phosphate synthase/phosphatase complex. Naturally, the 3' end of the otsB coding region overlaps the 5' end of the otsA coding region by 23 nucleotides. The Trehalose helps the frozen dehydrated cells to recover back to a functioning hydrated state. Not all cells survive the freeze drying process but Trehalose increases the rate of success.

 

Hydroxyectoine

The hydroxyectoine biosynthesis pathway present in Streptomyces Chrysomallus consists of four genes: thpA, thpB, thpC and thpD. In our system, we plan on The other three genes are there to initiate the process. When inserted into a bacterium, hydroxyectoine will prevent damage from dessication and heat.

Lyophilization

Lyophilization is the process by which water is extracted from our cells through sublimation. The process consists of first freezing the cells such that the water becomes ice, then placing them into a vacuum, causing sublimation of the ice into water vapor. Upon completion, you end up with a product shown above in the intro section. The process of freezing cells causes damage and can result in cell lysis. By including protectants such as trehalose, sucrose, and hydroxyectoine, this damage can be reduced and our Bactoblood can thus be stored for long periods of time.

The image above demonstrates trehalose's ability to stabilize the cell's lipid membrane and prevents damage upon rehydration. "Leakage" refers to the leakage of cell contents due to membrane damage, and it is prevented by incorporation of trehalose into the cell membrane.

References

P. Louis et al, "Survival of Escherichia coli during drying and storage in the presence of compatible solutes", Appl. Microbiol Biotechnol., 41:684-688, 1994

MAximino Manzanera et al., "High survival and stability rates of Escherichia coli dried in hydroxyectoine", FEMS Microbiology Letters, Vol. 223, Issue 2. pp. 347-352, 2004

Georg Lentzen and Thomas Schwarz, "Extremolytes: natural compounds from extremophiles for versatile applications", Applied Microbiology and Biotechnology, Vol 72, No. 4, 2006

Julia Prabhu et al., "Functional Expression of the Extoine Hydrozylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata", Applied and Environmental Mircobiology, vol. 70, No. 5, p. 3130-3132, 2004

SB Leslie et al, "Trehalose and sucrose protech both membranes and proteins in intact bacteria during drying", Appl. Environmental Biology, vol. 61, No. 10, 3592-3597, 1005

I Kaasen et al, "Molecular cloning and physical mapping of the otsBA genes, which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by katF (AppR), J. Bacteriology, 174(3): 889-898, 1992

Strom AR and Kaasen I, "Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression", Mol. Microbiol. 8(2): 205-10, 1993

W Boos et al. "Trehalose of Escherichia coli. Mapping and cloning of its structural gene and identification of the enzyme as a periplasmid protein induced under high osmolarity growth conditions.", J. Biol. Chem, Vol. 262, Issue 27, 13212-13218, 09, 1987.

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