We defined three main goals.
Express the antifreeze protein LpAFP in E. coli
Understanding freezing and antifreeze activity
Characterize Pseudoalteromonas nigrifaciens
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).
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.
The results reflect the importance of heterogeneous nucleation in the freezing of liquid water, this type of nucleation being a surface phenomenon . Besides, the results confirm that agitation induces the solidification of supercooled water, as this is an unstable liquid .
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.
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% . 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.
The PB and LB media and the tube with E. coli in LB medium were frozen at -5 °C after shaking.
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).
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 . 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 . Therefore, all the experiments we are conducting with this species will open a future line of research around this bacterium.
- 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.