Team:Tec-Monterrey/antifreeze/project

The antifreeze project aims to create a new bacterial strain that produces its own cryopreservant. Normally, as it is shown in the image, Escherichia coli is lysed when frozen, especially if this is done slowly. By adding a plasmid with a part that codes for an aniti-freeze protein and more parts that help to direct ths protein to the periplasm, we are conferring a freeze-thaw resistance to E.coli. This way, the ice crystals will stop their growth after freezing starts and the bacterium will not be lysed.

The antifreeze project aims to create a new bacterial strain that produces its own cryopreservant (anti-freeze protein). Normally, as it is shown in the image, Escherichia coli cells are lysed when frozen, especially if this is done slowly. By adding a plasmid with a part that codes for an anti-freeze protein and more parts that help to direct this protein to the periplasm and some of it to the cytoplasm, we are conferring a freeze-thaw resistance to E.coli. The protein makes the ice crystals stop their growth after freezing starts and prevents the bacterium from being lysed.

Cryopreservation is a technique that is used in laboratories worldwide1, to keep cells healthy and maintain in log phase growth for a long time. Nevertheless, this process is not 100% efficient because a portion of the cryopreserved cells are lost. To prevent these loses there are several cryoprotectants, like glycerol and DMSO, the most common cryoprotectant agents. Unfortunately, DMSO can cause some cells to differentiate and may be too toxic for others.2 Cryoprotectants has advantages and disadvantages; this is where anti-freeze proteins come into picture.

Antifreeze proteins (AFPs) play an important role in the resistance or tolerance to freezing of various organisms including fish, plants, and bacteria living in super-cooled conditions3; AFPs have diverse activities against the ice crystals. Their activity is related to their ability to bind to the surface of ice and inhibit its growth, causing a lowering of the freezing temperature; and another activity involves the ability to minimize the size of ice crystal and the rate of recrystallization. It has been found that AFPs from the beetle Rhagium inquisitor (RiAFP) have more antifreeze activity than other AFPs.4

The inevitable process of freezing and thawing can cause damage to the cells, these factors include length of storage between each successive freezing, initial cell concentration, suspending medium, and it is important for cells. We designed experiments that can analyze the interactions for different factors like temperature -80°C, -20°C and 4°C; different concentrations of proteins with the productions in different places (periplasm and citoplasm), different strains; and with different inductors (with arabinose and NaCl; with of arabinose and without inductors); all this interactions can give us information about better conditions.

1 Knight and Duman [Knight, C. A. & Duman, J. G. (1986) Cryobiology 23, 256-263] 2 Loewen MC, Liu X, Davies PL, Daugulis AJ.Biosynthetic production of type II Fish antifreeze protein: fermentation by Pichia pastoris 3 Properties, Potentials, and Prospects of Antifreeze Proteins 4 ARTHUR P. HARRISON, JR. SURVIVAL OF BACTERIA UPON REPEATED FREEZING AND THAWING'