Team:UPCH Peru/Poster

Poster: UPCH_Peru


CrioPROT: An innovative solution for crop loss due to frost


Team members
Jesús A. Durand Calle*, María I. Ruiz Ruiz*, Valeria A. Villar Dávila*, María C. Ortiz Cáceres*, María T. Castromonte Albinagorta*, Alfonso M. Rojas Montero*, Diego A. Benites Tan*, Oswaldo F. Lescano Osorio*, Rubén D. Velásquez Arbieto*, Elizabeth Sánchez Achulla**

Advisor
Ingrid L. Alarcón Ancajima***

Instructor
Nicolás Arias Vaccari*

PI
Daniel Guerra Giraldez*

* UPCH_Peru, Universidad Peruana Cayetano Heredia (UPCH)
** UPCH_Peru, Pontificia Universidad Católica del Perú (PUCP)
*** UPCH_Peru, Universidad Nacional Mayor de San Marcos (UNMSM)


In the Peruvian highlands, frosts during the winter cause crop damage leading to significant economic losses for small and medium scale farmers, perpetuating their already vulnerable condition. Our team wants to approach this problem by bringing an innovative solution.We aim to develop a system of production of an antifreeze agent which has a protective capacity in frost-susceptible crops, administrable by spraying. This product consists of a solution of recombinantly expressed and purified antifreeze proteins (AFPs). Our core genetic designs comprise the sequences of three types of AFPs, of plant and insect origin, with inducible and constitutive promoters, and a peptide signal for secretion to the culture medium. The chassis we have selected is a psychrophilic bacteria of the Pseudoalteromonas genus in order to guarantee an efficient work at low-temperature settings. In this way, our system will be capable of producing AFPs in low-tech environments in frost-affected regions.

Introduction

Frosts in Peru

Our country suffers from the consequences of a natural phenomenon called Frosts, in which the air temperature decreases below 0°C (32°F) or less1. The duration and frequency of this phenomenon (approximately 6 months) limits the plant growth affecting the agricultural sector in more than 60% of the Andean regions.

Moreover, frost damages have generated losses up to 180’000 hectares (1´800 km2) resulting in the peruvian agricultural communities being the most affected because agriculture represents the main livelihood of the small to medium farmers2. Their harvest is for self-consumption and for selling. If there are significant losses, they also lose a source of food and income, perpetuating their already vulnerable condition.


CrioPROT

Certain organisms use antifreeze proteins (AFPs) as a mechanism to survive in cold environments. These proteins bind to the ice crystals surface preventing ice crystal growth3.

We propose the development of an antifreeze product which will consist of a solution of recombinantly expressed and purified AFPs, and will be administered by spraying.


How did we do it?


To develop our project we have three specific aims:

  1. Selection and expression of AFPs.
  2. Understand freezing and antifreeze activity.
  3. Selection and characterization of a cold-tolerant organism.
Inspiration

One of our team members, Valeria Villar, told us about the problem and how it affects her family and community. Based on literature revision, we realized the magnitude of it and we knew we needed to do something about it.

1. Selection and expression of antifreeze proteins (AFPs)


We selected three AFPs: one from Tenebrio molitor (TmAFP4, 5) and two from perennial ryegrass Lolium perenne (LpAFP and LpIRI3)6, 7. At first, we designed their expression in E. coli (Fig. 1).

Figure 1. Genetic design for expression of AFPs in E. coli BLR.

After we cloned the composite part, we successfully induced the T7 promoter with IPTG to produce the antifreeze protein (Fig. 2).

Figure 2. Polyacrylamide gel stained with Commassie Blue showing AFP produced by induction in E. coli BLR (DE3) at 16°C. Soluble and insoluble fractions of the protein were extracted from bacteria at different hours after induction. Monomers of LpAFP can be seen at approximately 14kDa.
Engineering success

However, after the work done by Human Practices, we realized that the AFP production has to be made in low-tech environments such as the regions affected by frosts. We needed an organism able to thrive at temperatures below 0°C (32°F), so we finally selected Pseudoalteromonas nigrifaciens.

Consequently, we changed our former genetic design adding three new promoters from Pseudoalteromonas genus: one constitutive (Pasp8) and two inducible ones (pMAV9, pSHAB10); a P. nigrifaciens terminator (TaspC8) and a signal peptide for AFP secretion to the periplasmic space11(Fig. 3).

Figure 3. Genetic design for expression of AFPs in P. nigrifaciens.
2. Understand freezing and antifreeze activity


Working with AFPs demanded we understood the basis of how freezing occurs. Thus, we performed exploratory experiments to comprehend freezing and antifreezing activities.

First, we carried out freezing experiments with distilled water at subzero temperatures in the presence and absence of nucleating agents: plating beads, bond paper, and ground oregano (Fig. 4). We confirmed that the three materials function as nucleating agents at -5 °C; in contrast, water without any nucleating agent did not freeze even after shaking vigorously.

Figure 4. Test tubes with three different nucleating agents such as bond paper disc, ground oregano, plating beads (from left to right) in 5 mL of distilled water. Nucleating activity is checked.

Additionally, we carried out antifreezing experiments with aqueous solutions of chemical antifreeze agents (sodium chloride and glycerol in different concentrations) and of an heterogeneous nucleator (a plating bead) (Fig. 5). We observed that ice formed in the tubes with diluted concentrations of the antifreeze agent, but both sodium chloride and glycerol work effectively as antifreeze agents at a concentration of 3 Osmol/L.

Figure 5. Antifreeze activity of glicerol and sodium chloride at concentrations of 0.3 and 3 Osmol/L at -5°C.
3. Selection and characterization of a cold-tolerant organism


We seek these AFPs to be readily available in areas affected by frost on crops. Accordingly, our expression system needs to be able to work in cold temperatures; and for that we needed to test the viability of a candidate cold-tolerant organism. Based on literature revision, we choose Pseudoalteromonas nigrifaciens.

Engineering success

First, we determined and developed a suitable medium for the growth of P. nigrifaciens. We unsuccessfully tried to recreate the medium usually used for its growth. Later, based on the Pseudomonas bathycetes (PB) medium, we adapted the LB medium and P. nigrifaciens successfully grew on it (Fig. 6). Also, PB medium’s selectivity test showed only P.nigrifaciens growth (Fig. 7), which suggests that this medium is selective for this particular species.

Figure 6. Growth of P. nigrifaciens in PB medium.
Figure 7. Growth of P. nigrifaciens, E. coli, Pseudomonas spp., and Salmonella spp. in PB medium.

Moreover, we characterized morphology and bacterial growth. Staining characteristics confirm us that it is a gram-negative bacillus. Importantly, we observed that P. nigrifaciens is highly resistant to kanamycin and gentamicin but weakly resistant to spectinomycin, tetracycline and ampicillin. Its susceptibility to chloramphenicol was clear (Fig. 8). Finally, we established an appropriate electroporation protocol for P. nigrifaciens.

Figure 8. Growth curves of P. nigrifaciens in PB medium with different antibiotics at the same concentration. Control: no antibiotic.
Proposed implementation

The farmers affected by frosts will be the potential end users of our proposed product.


The crops affected by white frosts will be the type of frosts that could be treated with our product. The white frosts cause the formation of a layer of ice on the surface of plant leaves.



We defined that the product will be administered by spraying.

Need protein purification. Experimental procedures indicated that it will be necessary to purify the AFPs.

Why P. nigrifaciens?


We choose Pseudoalteromonas nigrifaciens, because:

  1. it has been used before as an expression system at low temperatures.
  2. is classified as a BSL-1 organism.
  3. can grow in conditions of high salinity, in which there is a low probability of contamination do not have many requirements for its maintenance.

Moreover, this bacteria has unique features like different types of promoters (constitutives and inducibles) which we could work with to produce the desired protein. Also, it has a periplasm in which proteins can be transported to, via a signal peptide specific of this bacteria.

Figure 9. Characteristic brown colonies of Pseudoalteromonas nigrifaciens.
Human Practices


Our team has been nourished by everything that this phenomenon implies on agricultural crops and the lives of farmers themselves. We have worked with key actors related to the issue of frosts in Peru by conducting surveys and interviews with specialist engineers on the subject and farmers from one of the most affected areas by this phenomenon. This information provides greater accuracy and validity to our project.

  • Key stakeholder interviews to:
    • Agricultural engineer William Carrasco Chilón, National Research Director of INIA (National Institute of Agricultural Innovation) in Cajamarca, Peru
    • Meteorological Engineer, Deputy Director of Weather Forecasting in SENAMHI (Peruvian National Service of Meteorology and Hydrology)
  • Activities with farmers from Junin, Peru

    • Main conclusions of:

Surveys

The participants agreed with the entry of science and technology into their working lives, they considered both necessary and precise an improvement in the face of the problems caused by frosts. They also agreed that our project would be beneficial and useful to fight this phenomenon.

Interviews

The participants not only suffered irreparable economic losses due to crop damage during frost season, but this also affected them both personally and emotionally.

Education

Being aware of the scarcely diffusion of science knowledge in our country, our team has focused on producing content related to science education. For that purpose, we organized three main activities.

We created a video series for the educational platform “Aprendo en Casa”, an effort of the Peruvian government for virtual learning during the COVID-19 pandemic. Based on the curricular program for basic education, we made videos about SynBio and its useful applications in approaching local problems, exemplified by our project.

Figure 10. Screenshots of the four videos elaborated for "Aprendo en Casa" program.

We organized an educational activity for high school students in which they were able to learn SynBio. The number of people registered were approximately 100 and were from many Peruvian regions.

Figure 11. Part of all the participants of our Education Activity.
Figure 12. Various regions of procedence of the participants.

We produced educational content to one of our stakeholders: the Andean farmers. We made conferences where we exchanged views about the problem of frosts to agriculture, and explained how biotechnology could be useful to overcome challenges of this nature.

Future plans

Due to the pandemic, we could not develop our project as originally planned, nevertheless, our commitment and work as a team in the future will be based mainly on:

  1. Proposing the expression of AFPs in P. nigrifaciens taking into consideration the cheaper way to obtain the product and according to the end user, the farmer. To do so, further steps should be:
    • Characterize our promoters.
    • Production and purification of the AFPs to compare their antifreeze activity.
    • Final production of our AFPs with Pseudoalteromonas bacteria.

  2. Maintaining permanent contact with farmers so that the project can satisfy real needs. We consider it necessary to implement it in projects that seek to respond to local problems.

We are opened to all experiments and possibilities to be explored.

References

  1. SENAMHI. (2010). Atlas de heladas del Perú. FAO - Organización de Las Naciones Unidas Para La Agricultura y La Alimentación, 50.
  2. Instituto Crecer. (2018). Del frío de la burocracia a las heladas de la Sierra | Blogs | NOTICIAS GESTIÓN PERÚ.
  3. 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.
  4. Yue CW, Zhang YZ. Cloning and expression of Tenebrio molitor antifreeze protein in Escherichia coli. Mol Biol Rep. 2009;36(3):529–36.
  5. Bar, M., Bar-Ziv, R., Scherf, T., & Fass, D. (2006). Efficient production of a folded and functional, highly disulfide-bonded β-helix antifreeze protein in bacteria. Protein Expression and Purification, 48(2), 243–252.
  6. Bredow, M., Vanderbeld, B., & Walker, V. K. (2017). Ice-binding proteins confer freezing tolerance in transgenic Arabidopsis thaliana. Plant Biotechnology Journal, 15(1), 68–81.
  7. Middleton, A. J., Marshall, C. B., Faucher, F., Bar-Dolev, M., Braslavsky, I., Campbell, R. L., Davies, P. L. (2012). Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site. Journal of Molecular Biology, 416(5), 713–724.
  8. Tutino, M. L., Parrilli, E., Giaquinto, L., Duilio, A., Sannia, G., Feller, G., & Marino, G. (2002). Secretion of alpha-Amylase from Pseudoalteromonas haloplanktis TAB23: Two Different Pathways in Different Hosts. Society, 184(20), 5814–5817.
  9. 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.
  10. 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.
  11. Giuliani M, Parrilli E, Sannino F, Apuzzo G, Marino G, Tutino ML. Soluble recombinant protein production in Pseudoalteromonas haloplanktis TAC125. Methods Mol Biol. 2015;1258:243–57.
Sponsors

We thank all our sponsors for their help.