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	<p>
<br></br>This project's objective is the development of a new <i>Escherichia coli </i>strain capable of surviving numerous steps of freezing and thawing, due to the expression of antifreeze proteins from the beetlle<i> Rhagium inquisitor</i> (RiAFP). We expect this new strain to be easier to handle in research labs and to increase the overall efficiency of transformed cells.</p>
	<p>We plan to modify this strain by transforming it with the necessary sequences for the expression of the RiAFP in the bacteria's cytoplasmic and periplasmic spaces. Furthermore, we will use an experimental design to find out the best storing conditions (inducer concentration, temperature and localization of the RiAFP) for this strain during cryopreservation.</p>
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<p><i>By enhahcing our E.coli with RiAFP, ice crsytals stop their growth and cell viability is preserved. The effect of RiAFP was assesed in periplams, and both periplasm and cytoplasm.  </i></p>

<br></br>
<p>Cryopreservation is a technique employed in laboratories worldwide to keep cells in log phase growth for a long time. Nevertheless, this process is not efficient because a significant portion of the cryopreserved cells are lost. To prevent these loses, cryoprotectans are added to the cells; such substances, however, have limited capacity in preventing damage from freezing. This is where anti-freeze proteins come into picture. The antifreeze project aims to create a new bacterial strain that produces its won cryopreservant by transforming with RiAFP. By creating a freeze resistant <i>E.coli</i> strain, laboratories around the world could use its properties as a complement to the other cryoprotectans and have a long lasting bacterial stocks with higher viability.
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<b>Background</b>
	<br></br>

	<p>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, <i>Escherichia coli </i> 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 <i>E.coli.</i> The protein makes the ice crystals stop their growth after freezing starts and prevents the bacterium from being lysed.  </p>
<p>
Cryopreservation is a technique that is used in laboratories worldwide (Knight et al,1986), 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 (Loewen et al, 1997). Cryoprotectants have advantages and disadvantages; this is where anti-freeze proteins come into picture.  </p>
<p>
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 conditions (Venketesh et all 2008); 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 <i>Rhagium inquisitor </i> (RiAFP) have more antifreeze activity than other AFPs (Harrison, 1955).
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.</p>
<p>
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.
</p>
<br></br>
<ul>
<li>1 Knight and Duman [Knight, C. A. & Duman, J. G. (1986) Cryobiology 23, 256-263]</li>
<li>2 Loewen MC, Liu X, Davies PL, Daugulis AJ.Biosynthetic production of type II Fish antifreeze protein: fermentation by Pichia pastoris </li>
<li>3 Properties, Potentials, and Prospects of Antifreeze Proteins</li>
<li>4 ARTHUR P. HARRISON, JR. SURVIVAL OF BACTERIA UPON REPEATED FREEZING AND THAWING'</li>
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