Team:UPCH Peru/Contribution


Application of AFP at iGEM in time

It is recently known that antifreeze proteins are useful for several fields besides the agricultural industry. That is why we considered that it will be relevant to make a compilation of the work done by previous iGEM teams in order to facilitate the next iGEM teams who want to use AFPs. In that sense, they could build their work from our revision and continue the spread of AFPs utilisation.


Team: WPI_Worcester

The WIP Worcester team was inspired by the 2018 romaine lettuce E. coli outbreak in the United States and the work of the 2015 WPI iGEM team. Therefore, the aim of this project is to apply effective antifreeze proteins to leafy crops to reduce the rate of foodborne illnesses caused by biofilm formation on leaves. They had to test the effect of various antifreeze proteins on biofilm formation and analyze the antibiofilm properties of ice structuring proteins (ISPs) and curcumin, a component of turmeric through E. coli strains that expressed antifreeze protein. Moreover, the biofilms were measured by the amount of extracellular polymeric substances (EPSs) they produced.

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The project arises from the problem of frost and high temperatures in the wine industry. To protect the vineyards, the team used a thermosensitive system to generate a protective effect, through the production of antifreeze proteins (against low temperatures) and reflective films (against high temperatures) by bacteria. In the first situation, some existing antifreeze proteins were improved, and efficiency tests were done. In the second case, efficiency tests were carried out with some reflective substances (caseins, chitin) to determine the best performance. With this they were able to build systems that, when they were at a low or high temperature, would produce the corresponding proteins.

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The Canadian project focuses on the issues of organ donation, transplant surgeries, and the variables involved in these procedures. Consequently, its main objective is improving the cryopreservation technique for organ storage. To do this, using type III antifreeze protein from the Zoarces americanus fish, they first sought to optimize antifreeze activity by adding multiple AFPs to a self-assembled protein scaffold that they named Ice Queen. Second, they decided to further explore the subject of AFPs and their applications, so they sought to increase the stability of the protein through circularization techniques.

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The project undertaken by the iGEM 2014 team from the Cambridge School of Weston (CSW) aims to design a synthetic biology solution to frost damage to crops based on the work of the 2009 Utah State team that developed a protein secretion mechanism and that of the 2011 Yale team that synthesized an "antifreeze" protein (RiAFP) isolated from a cold-tolerant beetle called the Rhagium inquisitor. According to this team, RiAFP is the most powerful antifreeze protein studied so far. They wanted to develop a device (Plantifreeze), which would be designed to mitigate the damage to crops caused by the formation of ice crystals on the surface of the plant. The difference with our project is that we want to design an easy to use product, like an AFP-based solution in spray, that the farmers can use in a self-sufficient way in their lands. Moreover, they use the antifreeze protein (RiAFP) and bacterium E. coli, our team will use Pseudoalteromona nigrifaciens.

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The Tec-Monterrey team focuses on the loss of cells in the cryopreservation technique. Their antifreeze project aims to create a new Escherichia coli strain capable of surviving numerous steps of freezing and thawing by producing its own cryopreservant. The modification of the strain is carried out through transforming it with the sequences for the expression of antifreeze proteins from the beetle Rhagium inquisitor (RiAFP). Moreover, they look for the best storing conditions for the strain during cryopreservation evaluating 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 cytoplasm), different strains; and with different inductors (with arabinose and NaCl; with of arabinose and without inductors).

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This team modified a bacteria in such a way that it can perform 2 different functions depending on the stimulus used that they called E.D. Frosti. The first function is to stimulate ice formation of ice crystals by producing Ice Nucleating Proteins (INP). They suggest that this function could have different applications, the decrease in ice-melting of glaciers is one of them. The second function of their creation is the anti-freeze function by the production of AntiFreeze Proteins (AFP). One of the applications that they proposed was the formation of an anti-freeze biofilm on the roads, which would help the roads become free of ice and snow in winters.

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The goal of this project was to demonstrate that Rhagium inquisitor antifreeze protein (RiAFP) is a protein with potential application in resistance to freezing or control of ice growth. They focused on a characterization study of this protein and its activity, since, to date, only its primary sequence and its thermal hysteresis activity had been determined. For this, they carried out cloning, expression, purification, function characterization, optimization and X-ray crystallography tests. The main difference with our project is that the use of this protein in a specific sector was not tested (as our group seeks to do in the field of agriculture).

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This project described the problem of crop loss due to frost, explaining that it occurred due to the formation of ice layers on the leaves that prevented gas exchange. It was concentrated in the Mexican coffee crops that are between 900 to 1800 meters above sea level. Their goal was to create a public domain cold-induced vector (as opposed to P. shock). They used “Cold Shock" mechanisms that prepare the bacteria to resist the cold by synthesizing proteins. This vector induces the expression of an antifreeze protein (AFP) obtained from a multiple alignment of 14 sequences from the Zoarces americanus fish, which was carried out to choose optimal codons for E. coli. The protein denatures at 37 ° C. Once you have enough protein, it is purified and applied to the leaves of the crops. Some differences with our project is the use of the protein-producing organism, they use E. coli and we use Pseudoalteromona nigrifaciens, which has shown ease to grow at low temperatures. We are looking to use an AFP from Lolium perenne, which is a plant. In addition, we seek to implement the use of home bioreactors so that the antifreeze can be produced and used by crop workers easily and without outside help.

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This team focused on terraforming Mars and, to do so, they have five conceivable key components which would play an important role to adapt to and modify the Martian environment.

-Iron-Oxidizing Bacteria
-Microbial Consortium
-Anti-Freezing Protein (AFP)
-Blackened Bacteria
-N2-Generating Bacteria

AFP is one of them because the surface temperature of Mars is below freezing point of water almost all day. The temperature doesn't allow most organisms to survive so they introduce AFPs into bacterias and confirm the expression of the AFP in the strain. The elected AFP was derived from mealworm and the bacteria was E.coli strains Origami 2.

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Does water freeze at 0°C?

Given that it was our first time doing freezing experiments, we considered it necessary to explore this phenomenon. In that sense, we performed the following experiments because it will be helpful for the next iGEM teams who want to explore it too. We detailed the methodology, results and discussion obtained with them.


While we explored numerous attempts to know at what temperature and in how long the freezing of distilled water occurs to corroborate that it's melting and a freezing point was 0 ºC (273.15 K) [1,2], we were surprised by that the water remained liquid at a temperature of -5 ºC, that is, without witnessing any physical change. However, when the mercury thermometer was introduced to measure the temperature of the medium and then stir it, the formation of small ice crystals was noted. In a matter of minutes, all the water had frozen.


The methodology applied for this trial was as follows:

  • 5 mL of distilled water was added to 6 test tubes and covered with a parafilm.
  • They were immersed in a recirculating 60% V/V ethanol bath.
  • The PolyScience recirculation equipment was programmed for the system to be at a temperature of -5 ºC.
  • Times of 30 min were taken from 1 hour to 3 hours.
  • The temperature of the distilled water inside the test tube was measured with the aid of a mercury thermometer.


The results obtained in this test are the following:

  • No ice formed in any of the test tubes at the temperature of -5 ° C.
  • The contact of the mercury thermometer with the wall of the test tube caused small ice crystals to appear.

* It should be noted that experiments were carried out previously with volumes of 100, 200, 300, 1000, and 1500 uL of distilled water using 1.5 mL microcentrifuge tubes and PCR; however, no ice formed in any of the tubes.


First of all, to understand this process of change of liquid state a solid, it must be taken into account that there are two mechanisms for freezing water: homogeneous and heterogeneous nucleation.

The phenomenon of homogeneous nucleation consists of the grouping of water molecules without the help of a nucleating agent [1,3]. Besides, it's characterized by its high activation energy and occurs spontaneously at temperatures close to -40 ºC [4,5]. It should be noted that liquid water in a range from 0 to -38 or -40 ºC is called supercooled water and this is characterized by presenting a metastable state [1,5]. On the other hand, when a nucleating agent "catalyst" is added to the solution for freezing to occur, it is designated heterogeneous nucleation. In that order of ideas, the nucleation of distilled water at -5 ºC does not occur because it does not present the necessary energy, since it is a slow process and requires a lower temperature, but when the thermometer has inserted the action of scraping or hitting the wall of the glass generated enough energy for nucleation to take place and therefore, the formation of the first ice crystals, which after a period of a few minutes grew due to the crystalline growth process.

*New* characterization of P. Nigrifaciens strain 217

  1. Characterization of the bacteria

    Since Pseudoalteromonas nigrifaciens is a bacteria that has not been widely used in the field of synthetic biology, we wanted to characterise deeper its growth and which factors would affect it. First, we did a bibliographic revision in which we could collect different aspects of our chassis. For it, we found the following information:

    This is a gram negative rod-shaped marine bacteria. It has a wide range of growth temperature, being inactivated at temperatures higher than 30°C - 37°C [7-9]. It is strictly aerobic and can survive to salt concentrations between 1% to 10% [8]. It can produce melanin pigmentation when growing at 4°C and could be inactivated if the medium contains ascorbic acid, EDTA or L-cysteine [9]. Does not need any additional growth factors. Also, it can metabolise citrate and sorbitol, has DNAse activity and motility due to its polar flagellum, among others [7-9]. Depending on the strain, its antibiotic susceptibility may vary. According to ATCC®, this bacteria is categorized as BSL-1. It also could grow at depth [6].

  2. Growth medium

    According to a few scientific articles characterising the Pseudoalteromonas genre[6,8,9], 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 simple medium in which our bacteria could grow optimally and be able to produce our protein in an efficient way. After trying to recreate this marine medium without success, due to the high amount of different salts needed and not efficient bacterial growth, we found another medium called Pseudomonas bathycetes medium that was suitable as well for P. nigrifaciens [10]. It consists of high concentrations of sodium chloride, proteose peptone, magnesium sulfate heptahydrate, magnesium chloride, yeast extract, and potassium chloride [10]. For its preparation, the composition of the LB medium was adapted and the additional salts concentrations were added. As it was prepared like broth and as a solid medium, it did not present precipitation even after these were autoclaved. After that, we proceeded to culture our bacteria by streak method, adding 20uL of the previous medium in which we activated our bacteria. Then, we let it grow again overnight 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 were clearer.

    We also could corroborate the selectivity of this medium through the culturing of three different bacteria: E. coli, Salmonella spp and Pseudomonas, besides P. nigrifaciens. As a result, the only bacteria that could survive at this high salt concentration was the latter. Thus, we could assure that this medium would avoid any possibility of accidental contamination.

  3. Electroporation protocol for P.nigrifaciens

    After reviewing the literature, we realized that the electroporation method, using the genus Pseudoalteromonas, has a low transformation efficiency (102 - 105 CFU / ug DNA) [11,14]. One of the main reasons is that this genus grows in high salt concentrations [15,16], which acts as a barrier, preventing the binding of exogenous DNA to the cell surface [16]. Furthermore, the abundant presence of restriction-modification systems, in the Pseudoalteromonas genome, decreases the efficiency of DNA internalization, since these systems degrade foreign DNAs [15-16]. On the other hand, containing multiple resistance genes and drug efflux pumps allows cells to survive antibiotic pressure; therefore, hindering the counter-selection of transformants [15-16].

    Likewise, contradictory results have been reported when handling strains of Pseudoalteromonas [16]. While in the study by Zhao et al. [13] it was possible to efficiently transform by electroporation the plasmid pWD2 into a strain of Pseudoalteromonas, Wang et al. [15] could not electroporate the same plasmid in other strains of the same genus. In contrast, in the latter study, the plasmid pWD2-oriT could be transferred by conjugation with Escherichia coli as donor cells [15]).

    Given that the DNA transferred by conjugation is single-stranded, unlike electroporation, this could reduce the possible degradation by restriction-modification systems that preferably degrade double-stranded DNAs [15,17]. As a result, conjugation techniques have been widely used for genetic manipulations in Gram negative bacteria, such as Pseudoalteromonas [15,18].

    However, the conjugation method is still time-consuming and certain problems can arise when selecting transconjugants after mating [16]. Therefore, we have chosen to transform P.nigrifaciens using electroporation. This species, specifically, does not have its own electroporation protocol, nor has it been transformed by any other method. However, other species and strains of Pseudoalteromonas have been used in electroporation and conjugation [11-15].

    Within the investigated electroporation protocols, we have chosen the one proposed by Delavat et al. [14], since it’s a rapid and efficient protocol to introduce exogenous DNA in Pseudoalteromonas sp. A small amount of plasmid DNA (200 ng) was enough to obtain 50-60 transformed colonies, which means that a transformation efficiency of at least 1.25 x 102 CFU / ug DNA was obtained [14]. Although this is not the highest efficiency, it remained constant when electroporating two different strains of Pseudoalteromonas sp., while other protocols only worked with one strain (6-8). This suggests that, by using Delavat’s protocol [14], we could obtain approximately the same efficiency by working with a different strain of the same genus (P. nigrifaciens strain 217). Furthermore, the protocol includes a wash buffer with 15% glycerol to avoid cell lysis, and allowing to quickly freeze competent cells in liquid nitrogen and store them at -80ºC until later use [14]. In the same article, it is mentioned that the cells kept full transformability after being stored for at least one month at -80ºC [14]. For these reasons, we expect that this protocol can be easily adapted to other marine strains such as P. nigrifaciens strain 217.

    Below is the adapted protocol [14] that describes the steps that should be followed to transform the plasmids in P.nigrifaciens:

    1.Cultivate the Pseudoalteromonas strain overnight at 24ºC.
    2.Place overnight cultures on ice and wash 2ml aliquots 3 times with 1 ml of an ice-cold modified sucrose buffer, containing 272 mM sucrose, 1 mM MgCl2,
    7 mM K2HPO4 adjusted to pH 7.5 and sterilized by autoclaving.
    3.Immediately before use, add 15% glycerol to the buffer to avoid cell lysis.
    4.After 3 washing steps, concentrate electrocompetent cells in 50uL with the same buffer and place the cells in an ice-cold 1 mm electroporation cuvette, mix them with 200 ng of plasmid DNA and directly electroporate them with a Gene Pulser II + Pulse Controller Plus (Biorad) set at 1.5 kV, 200 resistance and 25 F capacitance.

    5.Immediately after electroporation, add 1 ml of Pseudomonas bathycetes (PB) adapted medium and allow cells to recover for 4h at 24ºC.
    6.Plate cell suspensions on kanamycin plates and incubate for 2-3 days at 20ºC.


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