For our project, we addressed many problems related to our genetic design and the growth of our bacteria. In regard to our design, we researched and selected AFP of plant and insect origin as our antifreeze agent. Moreover, because of the views we received in our human practices activities, we had to adapt AFP production in a cold and low tech environment. In order to do that we made 7 new parts and put them in the Registry for AFP recombinant production in Pseudoalteromonas nigrifaciens.
In relation to our bacteria, selected for its efficient metabolism at low temperatures we selected Pseudoalteromonas nigrifaciens view. But this decision came with a challenge connected to the media in which our bacteria could be grown for our future experiments with AFP production. We decided not to opt for a commercially available media because we imagined a system that is viable in a low-tech environment for in-house production as our goal. To address this challenge, we researched and elaborated our own media.
Accordingly, we applied the engineering cycle in main aspects of our project:
In order to go about the problem of crop loss due to frost, we looked into state-of-the-art information regarding the frost season, and we summarized it in the following points:
Definition: Natural phenomenon characterized by air temperatures dropping below 0°C 
Occurrence: 6 month duration, being specially strong during 3 months 
Affected areas: Departments of the peruvian highlands 
General effects: Cattle raising and agricultural production are the most affected 
Effects in agriculture: It damages crops hindering their growth and production, affecting the national economy and food security 
Cellular mechanism for damage: Low temperature forms ice crystals in the intercellular spaces. As a result, water leaves the cells causing the formation of more ice crystals which will begin to stack in the intercellular spaces. Consequently, this has two effects: Mechanical damage and plant dehydration . More studies are needed to define where freezing begins: within or outside the plant. According to various studies, there are epiphyte nucleating bacteria (INA) that largely promote ice growth at the extracellular level 
This way, we learned this phenomenon greatly impacts agriculture, so we proceeded with learning about current solutions to this problem in our country. Literature search and the work done in Human Practices, we learned that traditional solutions demand a lot of time, money and work. Some of the current practices are:
Following a brainstorming session, it dawned on us that we could use characteristics from cold resistant organisms. This way, a literature search informed us that , unlike physiological mechanisms some possess, organisms like plants, insects and fish, produce antifreeze proteins (AFPs) . These AFPs work by binding on the surface of ice and, through two essential properties, diminish the freezing point and inhibit ice crystal growth. These two properties are thermal hysteresis (TH) and ice crystallization inhibition (IRI) .
We found that AFPs that have a high TH activity also have a low IRI activity, and vice versa. Thus, we decided to choose the extremes, meaning, those AFPs with a high activity of one of the properties and low activity in the other one . Therefore, we chose proteins from the perennial ryegrass Lolium perenne, LpAFP and LpIRI3, both of which have a high IRI activity [7,8]. Additionally, we chose a protein from Tenebrio molitor TmAFP, which is known for its high TH activity .
Thus, our first genetic designs contain only three AFPs (LpAFP, LpIRI3 and TmAFP) optimized for E. coli. We started our experiments by expressing LpAFP in E. coli. For that purpose we used our part BBa_K3612001 containing LpAFP gene and both T7 promoter and terminator. We successfully cloned and expressed the protein.
However, the work done by Human Practices made us realize that it will be necessary to produce AFP in low-tech environments, such as the regions affected by frosts. Given that these are characterized by low temperatures below 0°C (32°F), we needed an organism able to thrive in these conditions. We consulted the literature and found Pseudoalteromonas nigrifaciens, a biosafety level 1 marine bacteria. 
This bacteria has unique features like different types of promoters (constitutives and inducibles) which we could work with to produce the desired protein [11-14]. Also, it has a periplasm in which proteins can be transported to, via a signal peptide specific of the bacteria .
Consequently, we needed to change our former genetic design. The AFPs will be maintained but the sequence will be optimized for P. nigrifaciens. We added three new promoters: one constitutive called Pasp (part: BBa_K3612014) and two inducible called pMAV (part: BBa_K3612013) and pSHAB (BBa_K3612012). Moreover, we added a P. nigrifaciens terminator called TaspC (part: BBa_K3612011), and the signal peptide mentioned before for purification purposes (part: BBa_K3612015)
Further steps should be taken in order to: characterize our promoters, purify the protein itself and to produce it again in our Pseudoalteromonas bacteria.
For the protein
- Test the activity of LpAFP. Possibly by measuring ice recrystallization inhibition (IRI).
- Production of LpIRI3 and TmAFP.
- Purification essays of AFPs by columns.
- Evaluate differences in IRI activity between AFPs of plant origin (LpAFP and LpIRI3).
- Comparison of antifreeze activity between the three proteins.
For the promoters
- Construction of a reporters to characterize the promoters
- Evaluate the strength of the facultative promoters through time
For the bacteria
- Production of our AFPs with Pseudoalteromonas bacteria
We aim in the long run for choosing according to more developed criteria one design with a promoter and AFP convenient for escalation of production in Pseudoalteromonas. Also, we plan to make essays with plants to test antifreeze activity of the AFPs.
Developing an ideal medium for P. nigrifaciens
According to a few scientific articles characterising the Pseudoalteromonas genus-17], the most suitable culture medium for P. nigrifaciens was the Marine broth Difco® 2216 (BD 279110). However, for the purpose of our project, we needed to create a low-tech medium in which our bacteria could grow optimally and be able to produce our protein in an efficient way. Therefore, firstly we decided to recreate this medium with the elements we had at our laboratory. After the autoclaving process, we saw that few salts precipitated, possibly affecting the total salts concentration of the medium. Eitherway, we decided to continue the experiment and activated the bacteria. We put it in the incubator and let it grow for two days at 25°C degrees and luckily worked. Nonetheless, when we first cultured by streak method the solid medium did not seem to be the best option for our bacteria growth, not only due to the amount of different salts required to prepare it, but also because few colonies could grow in it, indicating that this was not a good nutritious source. We kept the plate at 4°C.
Hence, we researched other mediums with a high salt concentration that would be easier to recreate with few elements and could be rich in nutrients. Eventually, we found a medium preparation for a bacteria called Pseudomonas bathycetes that was suitable as well for P. nigrifaciens. It consists of high concentrations of sodium chloride, proteose peptone, magnesium sulfate heptahydrate, magnesium chloride, yeast extract, and potassium chloride. For its preparation, the composition of the LB medium was adapted and the additional salts concentrations were added. As it was prepared as a broth and as a solid medium, it did not present precipitation even after these were autoclaved. This was a good sign because the concentrations would not be altered. After that, we proceeded to culture our bacteria by streak method, adding 20uL of the previous medium in which we preserved our bacteria. Then, we let it grow again for two days at 25°C degrees. Compared to the first recreated medium, the new one had a major bacterial growth and the brownish colour of the colonies would be clearer.
To further investigate the differences between both mediums, we analysed the characteristics of the salts used in each case, including the calculation of their ionic strength.
It is observed that both media have similar ionic strengths (difference of 5.4%), considering only the saline species. This means that the total ion concentration in both media does not differ greatly, probably justifying why the medium with a lower diversity of salts is effective.
Finally, to corroborate that our adapted medium was selective for P. nigrifaciens, we decided to culture in the same medium three additional bacteria: E. coli, Salmonella spp and Pseudomonas. In the solid medium, we drawed separated streaks with each bacteria and let it grow in the incubator for two days at 25°C. The next day we only saw a brownish line in the middle of the medium that was made with P. nigrifaciens cells. Thus, we could assure that this medium would also act as a barrier for any possibility of accidental contamination.
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