iGEM 2020 — Team UPCH_Peru

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# Team:UPCH Peru/Notebook

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# Notebook

Our work of several weeks is organized on this page. As on the experiments page, it is divided by teams: synbio, characterization of *P. nigrifaciens* and freezing exploration. In each day you can visualize the activities that we carried out. Read them and see our work!

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# Team:UPCH Peru/Engineering

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* [Genetic Design](#Subtitle1)
* [*P. nigrifaciens*](#Subtitle2)
* [References](#Subtitle3)

# Engineering Success

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:

![](https://static.igem.org/mediawiki/2020/5/5f/T--UPCH_Peru--Engineering_GeneticDesignGA.png)

##### GENETIC DESIGN

[Know more](#Subtitle1)

![](https://static.igem.org/mediawiki/2020/d/d9/T--UPCH_Peru--Engineering_CultureMediaGA.jpeg)

##### CULTURE MEDIA

[Know more](#Subtitle2)

## Genetic Design

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 [[1]](#ref1)

- **Occurrence**: 6 month duration, being specially strong during 3 months [[1]](#ref1)
- **Affected areas**: Departments of the peruvian highlands [[1]](#ref1)
- **General effects**: Cattle raising and agricultural production are the most affected [[1]](#ref1)
- **Effects in agriculture**: It damages crops hindering their growth and production, affecting the national economy and food security [[2]](#ref2)
- **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 [[3]](#ref3). 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 [[4]](#ref4)

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:

- Scheduling the sowing time
- Sowing at slopes
- Covering crops with blankets
- Sowing at greenhouses

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) [[5]](#ref5). 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) [[6]](#ref6).

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 [[6]](#ref6). Therefore, we chose proteins from the perennial ryegrass Lolium perenne, LpAFP and LpIRI3, both of which have a high IRI activity [[7](#ref7),[8]](#ref8). Additionally, we chose a protein from *Tenebrio molitor* TmAFP, which is known for its high TH activity [[9]](#ref9).

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](http://parts.igem.org/Part%3ABBa_K3612001) containing LpAFP gene and both T7 promoter and terminator. We successfully cloned and expressed the protein.

[![](https://static.igem.org/mediawiki/2020/9/99/T--UPCH_Peru--Results_Molecular_Fig1_ColonyPCR-.png)](https://static.igem.org/mediawiki/2020/9/99/T--UPCH_Peru--Results_Molecular_Fig1_ColonyPCR-.png)[![](https://static.igem.org/mediawiki/2020/e/e9/T--UPCH_Peru--Results_Molecular_Fig2_Poliacrylamide_FINAL.png)](https://static.igem.org/mediawiki/2020/e/e9/T--UPCH_Peru--Results_Molecular_Fig2_Poliacrylamide_FINAL.png)

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. [[10]](#ref10)

This bacteria has unique features like different types of promoters (constitutives and inducibles) which we could work with to produce the desired protein [[11](#ref11)-[14]](#ref14). Also, it has a periplasm in which proteins can be transported to, via a signal peptide specific of the bacteria [[15]](#ref15).

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](http://parts.igem.org/Part%3ABBa_K3612014)) and two inducible called pMAV (part: [BBa\_K3612013](http://parts.igem.org/Part%3ABBa_K3612013)) and pSHAB ([BBa\_K3612012](http://parts.igem.org/Part%3ABBa_K3612012)). Moreover, we added a *P. nigrifaciens* terminator called TaspC (part: [BBa\_K3612011](http://parts.igem.org/Part%3ABBa_K3612011)), and the signal peptide mentioned before for purification purposes (part: [BBa\_K3612015](http://parts.igem.org/Part%3ABBa_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

1. Test the activity of LpAFP. Possibly by measuring ice recrystallization inhibition (IRI).
2. Production of LpIRI3 and TmAFP.
3. Purification essays of AFPs by columns.
4. Evaluate differences in IRI activity between AFPs of plant origin (LpAFP and LpIRI3).
5. Comparison of antifreeze activity between the three proteins.

For the promoters

1. Construction of a reporters to characterize the promoters
2. Evaluate the strength of the facultative promoters through time

For the bacteria

1. 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[[15]](#ref15)-[17]](#ref17), 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*[[18]](#ref18). 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.

[![](https://static.igem.org/mediawiki/2020/b/be/T--UPCH_Peru--Engineering_Micro_Figure1%262.png)](https://static.igem.org/mediawiki/2020/b/be/T--UPCH_Peru--Engineering_Micro_Figure1%262.png)

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.

[![](https://static.igem.org/mediawiki/2020/a/a4/T--UPCH_Peru--Engineering_Micro_Figure3.png)](https://static.igem.org/mediawiki/2020/a/a4/T--UPCH_Peru--Engineering_Micro_Figure3.png)

[![](https://static.igem.org/mediawiki/2020/4/43/T--UPCH_Peru--Engineering_Micro_Figure4.png)](https://static.igem.org/mediawiki/2020/4/43/T--UPCH_Peru--Engineering_Micro_Figure4.png)

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.

---

## References

1. SENAMHI. Atlas de heladas del Perú. FAO - Organización de las Naciones Unidas para la Agricultura y la Alimentación. 2010;50.
2. Instituto Crecer. Del frío de la burocracia a las heladas de la Sierra | Blogs | NOTICIAS GESTIÓN PERÚ. 2018
3. Wei C, Huang J, Wang X, Blackburn GA, Zhang Y, Wang S, & Mansaray LR. Hyperspectral characterization of freezing injury and its biochemical impacts in oilseed rape leaves. Remote Sensing of Environment, 2017,195, 56–66.
4. Hirano SS, Upper CD. [54] Bacterial nucleation of ice in plant leaves. In Methods in enzymology 1986 1, Vol. 127, pp. 730-738. Academic Press.
5. Chattopadhyay, MK. Antifreeze proteins of bacteria. Resonance, 2007, 12(12), 25–30.
6. Davies PL. Ice-binding proteins: a remarkable diversity of structures for stopping and starting ice growth. Trends in biochemical sciences. 2014 Nov 1;39(11):548-55.
7. Lauersen KJ, Brown A, Middleton A, Davies PL, Walker VK. Expression and characterization of an antifreeze protein from the perennial rye grass, Lolium perenne. Cryobiology. 2011;
8. Bredow, M., Vanderbeld, B., & Walker, V. K. Ice-binding proteins confer freezing tolerance in transgenic Arabidopsis thaliana. Plant Biotechnology Journal, 2017 15(1), 68–81.
9. Hakim A, Nguyen JB, Basu K, Zhu DF, Thakral D, Davies PL, Isaacs FJ, Modis Y, Meng W. Crystal structure of an insect antifreeze protein and its implications for ice binding. J Biol Chem. 2013 Apr 26;288(17):12295-304.
10. ATCC Products. Pseudoalteromonas nigrifaciens (ex White) Gauthier et al. (ATCC ® 19375). 2020.
11. Tutino ML, Parrilli E, Giaquinto L, Duilio A, Sannia G, Feller G, et al. Secretion of alpha-Amylase from *Pseudoalteromonas haloplanktis* TAB23: Two Different Pathways in Different Hosts. Society. 2002;184(20):5814–7.
12. Sannino F, Giuliani M, Salvatore U, Apuzzo GA, de Pascale D, Fani R, et al. A novel synthetic medium and expression system for subzero growth and recombinant protein production in *Pseudoalteromonas haloplanktis* TAC125. Appl Microbiol Biotechnol. 2017 Jan 27 ;101(2):725–34.
13. Papa R, Rippa V, Sannia G, Marino G, Duilio A. An effective cold inducible expression system developed in *Pseudoalteromonas haloplanktis* TAC125. J Biotechnol. 2007 Jan 1;127(2):199–210.
14. Cusano AM, Parrilli E, Marino G, Tutino ML. A novel genetic system for recombinant protein secretion in the Antarctic *Pseudoalteromonas haloplanktis* TAC125. 2006.
15. Ivanova EP, Kiprianova EP, Mikhailov VV et al. Phenotypic diversity of Pseudoalteromonas citrea from different marine habitats and emendation of description. Int J Syst Bacteriol, 1998, 48, 247-256. Available from: 10.1099/00207713-46-1-223
16. Nam YD, Chang HW, Park JR., et al. *Pseudoalteromonas marina sp. nov.*, a marine bacterium isolated from tidal flats of the Yellow Sea, and reclassification of Pseudoalteromonas sagamiensis as Algicola sagamiensis comb. nov. Int J Syst & Evol Microbiol, 2007, 57(1), 12–18. doi:10.1099/ijs.0.64523-0
17. Bowman, J. P., & McMeekin, T. A. (2015). Pseudoalteromonas. Bergey’s Manual of Systematics of Archaea and Bacteria, 1–22. doi:10.1002/9781118960608.gbm01098- Atlas R M.(2005) Handbook of Media for Environmental Microbiology. 2nd ed. CRC Press. p 656.

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# Team:UPCH Peru/Experiments

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# Experiments

On this page we detail the experiments we've carried out. They are separated by work teams: synbio, characterization of *P. nigrifaciens* and freezing exploration. Come and see them!

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# Team:UPCH Peru/Results

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  [Education & Engagement](/Team%3AUPCH_Peru/Education)

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* [Antifreeze protein expression](#Subtitle1)
* [Freezing and antifreeze](#Subtitle2)
* [Growth and characterization](#Subtitle3)
* [References](#Subtitle4)

# Results

#### We defined three main goals. - Express the antifreeze protein LpAFP in *E. coli* - Understanding freezing and antifreeze activity - Characterize *Pseudoalteromonas nigrifaciens*

## Antifreeze protein expression

[![](https://static.igem.org/mediawiki/2020/9/99/T--UPCH_Peru--Results_Molecular_Fig1_ColonyPCR-.png)](https://static.igem.org/mediawiki/2020/9/99/T--UPCH_Peru--Results_Molecular_Fig1_ColonyPCR-.png)

In order to test recombinant production of the AFPs we chose LpAFP for production in *E.coli* BLR(DE3) through expression vector pET28a. We made that decision because, according to literature, AFP of plant origin are easiest to produce than those from insects, and LpAFP, which was already properly characterized in literature, arrived first [[1](#ref1)-[3]](#ref3). Our PI kindly provided pET28 expressión vector that was of very common use in the lab and has sufficient expressión for a polyacrylamide gel visualization according to experience.

We assembled our vector with the LpAFP by double digestion with restriction enzymes and ligation with DNA ligase. Then, we transformed the product by electroporation in our *E.coli* BLR(DE3). We could evidence the presence of our genetic construct in the bacteria **(Figure 1)**

We induced protein production with IPTG , and extracted the product in a time-escalated series. We ran it through our polyacrylamide gel with Coomassie Blue staining. We could visualize very thin bands that matched the weight of the protein approximately 14 kDa **(Figure 2)** [[1](#ref1),[2]](#ref2). The results showed that we could produce recombinant antifreeze protein in our lab through *E. coli* BLR(DE3).

[![](https://static.igem.org/mediawiki/2020/e/e9/T--UPCH_Peru--Results_Molecular_Fig2_Poliacrylamide_FINAL.png)](https://static.igem.org/mediawiki/2020/e/e9/T--UPCH_Peru--Results_Molecular_Fig2_Poliacrylamide_FINAL.png)

---

## Understanding freezing and antifreeze activity

#### FREEZING EXPERIMENTS USING HETEROGENEOUS NUCLEATORS

The change of state from liquid water to ice involves two processes: nucleation and crystal growth. The first stage, nucleation, can be of two types: homogeneous and heterogeneous. The homogeneous one has elevated activation energy and occurs at temperatures below -40 °C. On the other hand, heterogeneous implies the formation of an ice core around the surface of a particle present in water (soluble or insoluble). The latter allows the freezing of liquid water to occur at higher temperatures and close to the theoretical freezing point (0 °C). The aim of this experiment is to determine the nucleating activity of the plating beads, bond paper, and oregano in a 5 mL sample of distilled water and at a temperature of -5 ° C.

#### **Results**- Plating beads, bond paper sheets, and oregano were found to function as nucleating agents. - It was found that the plating beads, in more than 1 hour, without agitation, promote nucleation. - In the test with oregano, no ice crystals form without shaking at -5 °C.

[![](https://static.igem.org/mediawiki/2020/c/ca/T--UPCH_Peru--Results_Metodo_Table1.png)](https://static.igem.org/mediawiki/2020/c/ca/T--UPCH_Peru--Results_Metodo_Table1.png)
[![](https://static.igem.org/mediawiki/2020/e/e5/T--UPCH_Peru--Results_Metodo_Figure1.png)](https://static.igem.org/mediawiki/2020/e/e5/T--UPCH_Peru--Results_Metodo_Figure1.png)
[![](https://static.igem.org/mediawiki/2020/c/c7/T--UPCH_Peru--Results_Metodo_Figure2.png)](https://static.igem.org/mediawiki/2020/c/c7/T--UPCH_Peru--Results_Metodo_Figure2.png)
[![](https://static.igem.org/mediawiki/2020/2/2b/T--UPCH_Peru--Results_Metodo_Table2.png)](https://static.igem.org/mediawiki/2020/2/2b/T--UPCH_Peru--Results_Metodo_Table2.png)

#### **Discussion**

The results reflect the importance of heterogeneous nucleation in the freezing of liquid water, this type of nucleation being a surface phenomenon [[4]](#ref4). Besides, the results confirm that agitation induces the solidification of supercooled water, as this is an unstable liquid [[5]](#ref5).

#### FREEZING EXPERIMENTS USING CHEMICAL ANTIFREEZE AGENTS AND HETEROGENEOUS NUCLEATORS

The freezing of liquid water is promoted in the presence of nucleating agents by the phenomenon of heterogeneous nucleation. The aim of this experiment is to compare the antifreeze activity of NaCl and glycerol in different concentrations and in the presence of an heterogeneous nucleator.

#### **Results**- Ice formed in the tubes with diluted concentrations of antifreeze 0.3, 0.4, 0.5, and 0.6 Osmol/L. - The antifreeze activity of glycerol and sodium chloride are effective at 3 Osmol/L.

[![](https://static.igem.org/mediawiki/2020/0/06/T--UPCH_Peru--Results_Metodo_Figure3.png)](https://static.igem.org/mediawiki/2020/0/06/T--UPCH_Peru--Results_Metodo_Figure3.png)
[![](https://static.igem.org/mediawiki/2020/a/a4/T--UPCH_Peru--Results_Metodo_Figure4.png)](https://static.igem.org/mediawiki/2020/a/a4/T--UPCH_Peru--Results_Metodo_Figure4.png)
[![](https://static.igem.org/mediawiki/2020/c/c7/T--UPCH_Peru--Results_Metodo_Table3.png)](https://static.igem.org/mediawiki/2020/c/c7/T--UPCH_Peru--Results_Metodo_Table3.png)

#### **Discussion**

Sodium chloride can lower the freezing point of water by 21.0 °C at a concentration of 23%, while glycerol lowers it by 22.0 °C at a concentration of 50% [[6]](#ref6). Results corroborate the antifreeze capacity of both substances, even in the presence of a nucleating particle and with agitation. This suggests that an antifreeze agent may be more effective in its competition with a nucleator depending on its concentration.

#### FREEZING EXPERIMENTS WITH CULTURE MEDIA

The aim of this experiment is to evaluate the nucleating or antifreeze ability at -5 °C of the culture media used in microbiological experiments: a salty medium for *P. bathycetes* (PB) and Luria-Bertani medium (LB) with and without *E. coli*.

#### **Results**

The PB and LB media and the tube with *E. coli* in LB medium were frozen at -5 °C after shaking.

[![](https://static.igem.org/mediawiki/2020/6/64/T--UPCH_Peru--Results_Metodo_Figure5.png)](https://static.igem.org/mediawiki/2020/6/64/T--UPCH_Peru--Results_Metodo_Figure5.png)
[![](https://static.igem.org/mediawiki/2020/4/4d/T--UPCH_Peru--Results_Metodo_Table4.png)](https://static.igem.org/mediawiki/2020/4/4d/T--UPCH_Peru--Results_Metodo_Table4.png)

#### **Discussion**

The results show that the culture media of interest for microbiological experiments act as nucleating agents and not as antifreeze agents. These suggest that the expressed IBPs, before being used for agricultural purposes, should be purified since it would suppose an additional competence to prevent freezing.

---

## Growth and characterization of *Pseudoalteromonas nigrifaciens* strain 217

*P. nigrifaciens* can either grow in PB or Marine medium at 25°C, but the former showed optimal growth **(Figure 1)**. Staining characteristics confirm that it is a gram-negative bacillus **(Figure 2)**. PB medium’s selectivity test showed only *P. nigrifaciens* growth **(Figure 3)**, which suggests that this medium is selective for this particular species. We observed that strain 217 is highly resistant to kanamycin and gentamicin but weakly resistant to spectinomycin, tetracycline and ampicillin **(Figure 4)**. Its susceptibility to chloramphenicol was clear **(Figure 4)**.

[![](https://static.igem.org/mediawiki/2020/a/ad/T--UPCH_Peru--Results_Micro_Figure1.png)](https://static.igem.org/mediawiki/2020/a/ad/T--UPCH_Peru--Results_Micro_Figure1.png)
![](https://static.igem.org/mediawiki/2020/7/72/T--UPCH_Peru--Results_Micro_Figure2.png)
[![](https://static.igem.org/mediawiki/2020/6/6c/T--UPCH_Peru--Results_Micro_Figure3.png)](https://static.igem.org/mediawiki/2020/6/6c/T--UPCH_Peru--Results_Micro_Figure3.png)
[![](https://static.igem.org/mediawiki/2020/c/c3/T--UPCH_Peru--Results_Micro_Figure4.png)](https://static.igem.org/mediawiki/2020/c/c3/T--UPCH_Peru--Results_Micro_Figure4.png)

So far, we have successfully characterized the bacterium that will be used to produce the antifreeze proteins. We determined that the PB medium allowed better bacterial growth compared to the Marine Medium used in other studies **(Figure 1)** [[7](#ref7)-[9]](#ref9). Regarding antibiotic susceptibility, we determined that *P. nigrifaciens* strain 217 is highly resistant to kanamycin and gentamicin, which differs from what was found in other strains of this bacterium [[10]](#ref10). This confirms that the strain of *P. nigrifaciens* we plan to use for the production of the antifreeze proteins is likely to be different from strains previously studied.

The characterization we have made of *P.nigrifaciens* helps us continue with the transformation and AFP expression experiments. Although *Pseudoalteromonas* genus has been widely used for recombinant protein expression [[11](#ref11)-[16]](#ref16), *P. nigrifaciens*, particularly, has been scarcely explored at a morphological and genetic level [[17]](#ref17). Therefore, all the experiments we are conducting with this species will open a future line of research around this bacterium.

---

## References

1. Lauersen KJ, Brown A, Middleton A, Davies PL, Walker VK. Expression and characterization of an antifreeze protein from the perennial rye grass, Lolium perenne. Cryobiology. 2011 Jun;62(3):194-201. doi: 10.1016/j.cryobiol.2011.03.003. Epub 2011 Mar 30. PMID: 21457707.- Middleton AJ, Marshall CB, Faucher F, Bar-Dolev M, Braslavsky I, Campbell RL, Walker VK, Davies PL. Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site. J Mol Biol. 2012 Mar 9;416(5):713-24. doi: 10.1016/j.jmb.2012.01.032. Epub 2012 Jan 28. PMID: 22306740.
   - Hakim A, Nguyen JB, Basu K, Zhu DF, Thakral D, Davies PL, Isaacs FJ, Modis Y, Meng W. Crystal structure of an insect antifreeze protein and its implications for ice binding. J Biol Chem. 2013 Apr 26;288(17):12295-304. doi: 10.1074/jbc.M113.450973. Epub 2013 Mar 12. PMID: 23486477; PMCID: PMC3636913.
   - Kanji ZA, Ladino LA, Wex H, Boose Y, Burkert-Kohn M, Cziczo DJ, Krämer M. Overview of ice nucleating particles. Meteorological Monographs. 2017 Ene 1;58:1.1-1.33.
   - Chang R, Goldsby KA. Química. 12th ed. México, D.F.: McGraw-Hill Education; 2017. p. 501.
   - Fink JK. Antifreeze agents. En Fink JK. Petroleum engineer’s guide to oil field chemicals and fluids. USA: Gulf Professional Publishing; 2012. p. 445-453.
   - Ivanova EP, Kiprianova EA, Mikhailov V V., Levanova GF, Garagulya AD, Gorshkova NM, et al. Characterization and identification of marine *Alteromonas nigrifaciens* strains and emendation of the description. Int J Syst Bacteriol. 1996 Jan 1;46(1):223–8.
   - Ivanova EP, Kiprianova EA, Mikhailov V V., Levanova GF, Garagulya AD, Gorshkova NM, et al. Phenotypic diversity of *Pseudoalteromonas citrea* from different marine habitats and emendation of the description. Int J Syst Bacteriol. 1998;48(1):247–56.
   - Ivanova EP, Matte GR, Matte MH, Coenye T, Huq A, Colwell RR. Characterization of *Pseudoalteromonas citrea* and *P. nigrifaciens* Isolated from Different Ecological Habitats Based on REP-PCR Genomic Fingerprints. Syst Appl Microbiol. 2002 Jan 1;25(2):275–83.
   - Gorshkova NM, Ivanova EP. Antibiotic Susceptibility as a Taxonomic Characteristic of Proteobacteria of the Genera Alteromonas, Pseudoalteromonas, Marinomonas, and Marinobacter. Russ J Mar Biol. 2001;27(2):116–20.
   - Duilio A, Tutino ML, Marino G. Recombinant protein production in Antarctic Gram-negative bacteria. Methods Mol Biol. 2004;267:225–37.
   - Giuliani M, Parrilli E, Tutino ML, Sannia G, Marino G. Novel expression systems for recombinant protein production at low temperatures [Internet]. 2004. p. 1–8.
   - Médigue C, Krin E, Pascal G, Barbe V, Bernsel A, Bertin PN, et al. Coping with cold: The genome of the versatile marine Antarctica bacterium *Pseudoalteromonas haloplanktis* TAC125. Genome Res. 2005 Oct;15(10):1325–35.
   - Parrilli E, Duilio A, Tutino ML. Heterologous protein expression in psychrophilic hosts. In: Psychrophiles: From Biodiversity to Biotechnology. 1st ed. Berlin: Springer Berlin Heidelberg; 2008. p. 365–79.
   - Wilmes B, Hartung A, Lalk M, Liebeke M, Schweder T, Neubauer P. Fed-batch process for the psychrotolerant marine bacterium *Pseudoalteromonas haloplanktis*. Microb Cell Fact. 2010 Sep 21;9(1):72.
   - Wang P, Yu Z, Li B, Cai X, Zeng Z, Chen X, et al. Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas. Microb Cell Fact. 2015 Jan 23;14(1):11.
   - Baumann P, Baumann L, Bowditch RD, Beaman B. Taxonomy of Alteromonas: *A. nigrifaciens* sp. nov., nom. rev.; A macleodii; and A. Haloplanktis. Int J Syst Bacteriol. 1984 Apr 1;34(2):145–9.

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