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Applications

Our project is fundamental in nature. However, some promising applications for our project do exist. One of these applications is the usage of cold resistance as a selection method instead of antibiotics. The more obvious application is the ability to grow bacteria, or other organisms, at lower temperatures. This could be useful for the production of biofuels with a better efficiency. Finally, other iGEM teams could also benefit from our CryoBricks.

Biosynthesis

We consider biosynthesis -- that is, the production of various biological compounds through cell factories -- to be an area of interest, where our project can find many useful applications. Equipping cell factories with CryoBricks and successfully culturing them at lower temperatures may increase their efficiency in three different ways. These relate to the effect of temperature on three different levels: whole cells, enzymes, and the product. Note that while the effect of lowering the temperature can be divided into these levels, actually lowering this temperature is facilitated on the enzyme level, by the proteins encoded in our CryoBricks.

The first level is that of the population and whole cells. Addition of a plasmid containing our CryoBricks may allow cell factories to grow faster at temperatures below their optimum. This may reduce the costs of a production process by saving energy on heating, or it may increase the yield if culturing the cell factories at their optimal growth temperature isn't an option for whatever reason.

The second level is the enzyme level. Certain heat sensitive enzymes can function better at lower temperatures. If a cell factory comprises heat sensitive enzymes, lowering the temperature may increase their kinetic activity. In extreme cases, temperature governs not just the kinetic activity but also the solubility of the enzymes. If the heat sensitive enzymes are involved with the production of the compound of interest, lower temperatures may directly increase its rate of formation or its yield. Alternatively, if the enzyme is involved with cell maintenance or growth, product yield or formation speed may still be influenced indirectly by facilitating a higher cell density or growth rate.

The third scale is that of the product: the biological compound the cell factory is used to produce. Certain products may be unstable, and dropping the temperature can inhibit their rate of degradation by simple thermodynamics. If the product is actively degraded, there is also a chance the decrease in temperature slows down the enzymes that would achieve this. Note that if the product is an enzyme rather than a metabolite, as is the case with many cell factories, thermodynamic degradation won't play much of a role, but inhibiting the proteases that would break them down is all the more valuable.

Biofuel Production

Biofuels are expensive. According to the U.S. Department of Energy's Aquatic Species Program, biodiesel production costs are projected to be at least twice as high as the price of ordinary petroleum diesel, even with very aggressive assumptions about bioproductivity [http://www.nrel.gov/docs/legosti/fy98/24190.pdf [1]]. Currently, over 90% of commercial algae biomass production is done with large-scale open pond systems. There are no feasible alternatives to this setup, because of the high pressure on cost efficiency. Unfortunately, such systems usually suffer from low production rates during the cold season [http://www.nrel.gov/docs/legosti/fy98/24190.pdf ][http://www.futureenergyevents.com/algae/whitepaper/algae_positionpaper.pdf]. Heating the ponds is not an option either, because the energy required for this would cost more than the gained yield brings in.

Following the publication that cyanobacterial strains isolated from high latitudes often remain active at low temperatures [http://www.ffgg.ulaval.ca/paleoecologie/wp-content/uploads/2011/06/Tang.1997.pdf], Hong et al. (2010) identified the opportunity for cold-resistant cyanobacteria to enhance the efficiency of biofuel synthesis.[http://www.e-algae.kr/Upload/files/ALGAE/PW%EC%B5%9C%EC%A2%85_no6_Alagae25%282%29_10-10up.pdf] If our CryoBricks can be expressed in species other than E. coli, our project might be a humble first step down this road Hong et al. point towards.

Selection

An antibiotic is a compound or substance that slows down growth or kills bacteria. Bacteria can gain resistance for antibiotics by natural selection; if one micro-organism gains this ability it can share this ability through horizontal gene transfer. Although antibiotic resistance is very dangerous for human health and preventing the spread of antibiotics is top priority in hospitals and other medical facilities, selection of bacteria on antibiotic resistance is an essential tool in research.

When genes are introduced in bacteria, a plasmid is inserted into the bacteria containing the gene of interest along with a gene that grants antibiotic resistance. By adding antibiotics to the medium or plate, only bacteria that posess the resistance and your gene of interest can grow.

Our bricks will allow bacteria to grow in a cold environment. If the antibiotic resistance gene is replaced by one of our genes you can simply expose the medium or plate to the cold and only the bacteria that posses the cold resistance will grow.

This could be useful as alternative selecting method if you need a lot of different selection conditions, because it needs to contain different plasmids, or as new selection method because you do not want to use expensive antibiotics. Reasearch laboratoeries use a lot of antibiotics as selection method. An additional advantage of this method is that it eliminates some of the antibiotics that get flushed down the drain and end up in the environment.

iGEM teams

Growing bacteria under lower temperatures can potentially help a lot of other iGEM teams. This is most relevant to teams who have a project that intends to intorduce bacteria to inhospitable conditions. Here we present a selection of teams that caught our eye.

TU Delft 2010

The TU Delft 2010 team attempted to design an E.coli that was able to remove oil from the ocean after an oil spill. For their E.coli to work optimally it was engineered to survive in saltwater since E.coli cannot normally cope with high salt concentrations. They did not, however, prepare their bacteria for the cold temperatures present in open water. Please note that their team did not intend to release their bacteria into the envornment. They imagined a large tank containing their bacteria to be punped full of contaminated water where it was rinsed, and then filtering out the bacteria. In their original design the saltwater would have to be warmed before the bacteria could efficiently perform their function, but our CryoBricks coud eliminate the need for this.

Brown-Stanford 2011

This year’s Brown-Stanford team is attempting to use synthetic biology to allow bacteria to survive on Mars. Since Mars is a very cold planet, the availability of bacteria able to survive at subzero temperatures would allow one of the hurdles presented in this inhospitable environment.

References

1. Sheehan et al. A Look Back at the U.S. Department of Energy’s Aquatic Species Program — Biodiesel from Algae Executive Summary, page 2 (1998)
2. Benemann Opportunities and challenges in algae biofuels production (2008)
3. Tang et al. Cyanobacterial dominance of polar freshwater ecosystems J. Phycol. 33, 171-181 (1997)
4. Hong et al. Axenic purification and cultivation of an Arctic cyanobacterium Algae 25, 99-104 (2010)

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