Team:UPCH Peru/Results

• Antifreeze protein expression
• Freezing and antifreeze
• Growth and characterization
• References

Results

Antifreeze protein expression

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 ^[1-3]. 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) ^[1,2]. The results showed that we could produce recombinant antifreeze protein in our lab through E. coli BLR(DE3).

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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.

Discussion

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

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.

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]. 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.

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.

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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).

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) ^[7-9]. 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]. 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 ^[11-16], P. nigrifaciens, particularly, has been scarcely explored at a morphological and genetic level ^[17]. Therefore, all the experiments we are conducting with this species will open a future line of research around this bacterium.

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References

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2. 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.
3. 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.
4. 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.
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