Yinmeng Li, Alex Tang and Siwen Yang
Western Reserve Academy, Hudson, Ohio, USA
Reviewed on 8 May 2021; Accepted on 28 June 2021; Published on 25 October 2021
With help from the 2021 BioTreks Production Team.
Keywords: Ice nucleation, Pseudoalteromonas haloplanktis, global climate change, psychrophiles, lnaZ
Authors are listed in alphabetical order. Beth Pethel mentored the group. Please direct all correspondence to email@example.com.
The Arctic sea ice moderates the global climate by reflecting the sunlight back into space. However, the increased greenhouse gas emission has caused rapid loss of Arctic ice, in turn resulting in more heat being absorbed by the ocean surface. Starting from 1980, the Arctic ice sheets have been retreating at a rate of 2.6 percent per decade (National Snow & Ice Data Center, 2021), and more rapid ice loss is projected to be underway (NASA’s Earth Science News Team, 2020). As temperatures rise, ice sheets will melt faster than they can form new ones, resulting in a rise in global sea levels. In order to slow the loss of Arctic ice, our team proposes a design that utilizes Pseudomonas syringae and Marinomonas primoryensis.
P. syringae is a bacterium that induces frost damage in plants. The gene INA, or inaZ, codes for the ice nucleation proteins, which P. syringae uses to cause frost injuries to plants and gain entry into the plant tissue (Xin et al., 2018). The gene inaZ includes a transport sequence that anchors the ice nucleation proteins on the surface of P. syringae (Kassmannhuber et al., 2020), which prompts the crystallization of ice when in contact with water molecules. Ice nucleation proteins induce the formation of ice with a planar arrangement of hydrogen binding groups that complements the arrangement of an ice crystal surface. When linked together, the hydrogen binding groups can induce massive amounts of ice formation (Sherman & Lindow, 1993).
We plan to transform M. primoryensis with the gene inaZ. M. primoryensis was first isolated from coastal sea-ice (Romanenko et al., 2003) and can be cultivated on Marine Agar at up to 20°C (Vance et al., 2018). Being a psychrophile, it is capable of surviving in extremely low temperatures. The organism also has a tendency to bind to ice, which will allow the recombinant strain to create ice crystals around existing sheets of ice. However, there have been no documented transformations of M. primoryensis, and the feasibility of various transformation methods remain unknown. Hence, we plan to transform Pseudoalteromonas haloplanktis as the prototype of our design since it has been successfully transformed to secrete ɑ-amylase through interspecies conjugation (Tutino et al., 2002). P. haloplanktis is a psychrophilic bacterium typically cultured in YTP media at 4℃ (Parrilli, 2008).
The final rendition of our project will create a recombinant M. primoryensis strain transformed with the inaZ gene. As it naturally binds to ice, the ice nucleation proteins coded by the inaZ gene allows the transformed M. primoryensis to promote ice crystallization around existing ice sheets.
Our initial design is to engineer the gene inaZ into M. primoryensis, an existing bacterium in the Arctic ice (see Figure 1). However, since there have been no documented successful transformations of M. primoryensis, we plan to implement the design, instead, by inserting inaZ into P. haloplanktis TAC125, one of the most well-researched psychrophiles with previous cases of successful transformation (Duilio et al., 2004), through interspecific conjugation. P. haloplanktis presents a high level of similarities with the originally targeted organism, M. primoryensis, as both are gram-negative bacteria isolated from Antarctic coastal seawater.
P. haloplanktis will be transformed through interspecies conjugation, which requires transformed Escherichia coli cells that express the inaZ gene. Similar to P. syringae, the recombinant P. haloplanktis TAC125 should display ice nucleation proteins on its outer membrane (Kassmannhuber et al., 2020). Upon confirming the efficacy of our design, we will begin experimentation with M. primoryensis. While attaching itself to ice, the ice nucleation proteins on the outer membrane of recombinant M. primoryensis will promote ice nucleation at a higher temperature. Both the recombinant P. haloplanktis and M. primoryensis will be tested for efficacy with a droplet-freezing assay of ultra-pure water (Kassmannhuber et al., 2017). An aliquot of the transformed bacterial culture, mixed with ultra-pure water, will be cooled in a partially submerged well plate with an ethanol bath. Light is transmitted through the aliquot and its intensity, which changes upon freezing, is monitored by a camera. This method allows for the detection of freezing temperatures (David et al. 2019).
The inaZ gene is responsible for the expression of ice nucleation proteins. In P. syringae, the ice nucleation proteins are arranged and anchored on its outer cell membrane (Lindow et al., 1982). When in contact with water, the ice nucleation proteins arrange water molecules into ice-like structures (Kassmannhuber et al., 2017), allowing the formation of ice.
The inaZ gene will be synthesized along with corresponding restriction sites and activated by the promoter PhaspC. Both the promoter PhaspC and the terminator Tasp have been shown to function in psychrophiles after interspecies conjugation (Tutino et al., 2002). The inaZ gene will be cloned into pFF, a plasmid capable of replication in both E. coli and psychrophilic bacteria. The vector will be first inserted into P. haloplanktis TAC125 via interspecies conjugation with transformed E. coli (Tutino et al., 2002). If the ice nucleation protein is successfully expressed in P. haloplanktis, M. primoryensis will be transformed with the same plasmid and procedures (Figure 2).
Constructed from the pUCL vector (modified from pUC18 by removing the restriction site NdeI, a promoter, and the lacI gene), the pFF contains OriR (cold-adapted origin of replication) and OriT (origin of conjugative transfer), enabling it to function within E. coli and psychrophiles through conjugation (Tutino et al., 2002). The pFF vector （Figure 3) also possesses the PhaspC promoter and the Tasp terminator, both of which have been proven to function within psychrophiles such as P. haloplanktis in low temperatures. The inaZ gene will be synthesized along with a restriction enzyme and cloned into the pFF plasmid utilizing the restriction site EcoRI (see Figure 4). The EcoRI restriction site is found between the Pasp promoter and the Tasp terminator in the pFF plasmid and produces an overhang, ensuring that the inaZ gene will be ligated in the correct orientation. Since the sequence of the pFF plasmid is currently unknown, a restriction digest will be performed with EcoRI to ensure only one EcoRI restriction site is present within the plasmid. The pFF plasmid is chosen despite its unclear sequence because it has been shown to function effectively within psychrophiles such as P. haloplanktis, and is compatible with interspecies conjugation (Tutino et al., 2002).
|Figure 3. The plasmid structure. The pFF plasmid is compatible with interspecies conjugation.|
|Figure 4. Diagram of biological parts. The inaZ gene, coding for the production of the ice nucleation proteins, will be controlled by the PhaspC promoter (also notated as Pasp) and the Tasp terminator once cloned into the pFF vector.|
Both M. primoryensis and P. haloplanktis are bacteria native to Arctic regions. Since the only change in the recombinant M. primoryensis is the addition of the ice nucleation protein, we anticipate no negative effect on the Arctic ecosystem. Future experimentations with recombinant strains will be carried out to ensure the insertion of inaZ does not interfere with metabolic pathways of the bacteria. P. haloplanktis produces Volatile Organic Compounds that suppress the growth of Burkholderia cepacia complex strains (Sannino et al., 2016), pathogenic strains of bacillus present within the water, soil, and animal surfaces (Zhang & Xie, 2007). The recombinant P. haloplanktis will be deployed into seawater and should have no effect on the native Arctic Burkholderia genera, as they are commonly found within heathland (Hill et al., 2015) and tundra soils (Tao et al., 2020). We realize that the organism will need to be fully vetted before it is released into the environment and will conduct experiments and simulations to determine its exact effects over the environment.
P. primoryensis and P. haloplanktis have been cultivated under lab conditions (Vance et al., 2018). They are not pathogens and are safe to grow.
We chose M. primoryensis as our chassis because of its ability to bind to ice. Ideally, the recombinant M. primoryensis would allow the formation of ice on existing ice sheets. However, M. primoryensis has not been well-researched, hence the results of the transformation are relatively unpredictable. The presence of ice-binding proteins within the organism might also affect the efficacy of our design, as they have been shown to demonstrate ice interactivity, influencing ice crystal growth and shaping the ice morphology (Delesky 2021). Ice binding proteins are typically present within the organism to prevent freezing of their body fluids (Dolev et al., 2016), but we are currently unsure of its influence over our design. The recombinant M. primoryensis will be tested by a droplet-freezing assay. If the recombinant organism does not show significant ice nucleating abilities due to the interference of the ice-binding proteins, P. haloplanktis TAC125 will be employed as the chassis for the final design.
Our design currently remains in the theoretical phase. For further development of the design, the gene inaZ will be cloned into the pFF vector, which will be used for the subsequent transformation of E. coli. P. haloplanktis and M. primoryensis will then be transformed with recombinant E. coli through interspecies conjugation.
The prototype of the design was proposed by A.T. and improved upon by Y.L., A.T. and S.Y. Y.L. produced all graphics in this article; A.T. produced the video.
The Western Reserve Academy synthetic biology team would like to thank BioRender.com for their assistance in creating the figures and Dr. Beth Pethel for her guidance over the design.
National Snow & Ice Data Center. (2021, Apr 6). Arctic Sea Ice News & Analysis. Retrieved Apr 27, 2021, from http://nsidc.org/arcticseaicenews/
David, R. O., Castresana, M. C., Brennan, K. P., Rösch, M., Els, N., Werz, J., Weichlinger, V.,
Boynton, L. S., Bogler, S., Dedekind, N. B., Marcolli, C., & Kanji, Z. A. (2019, July).
Development of the DRoplet Ice Nuclei Counter Zürich (DRINCZ): Validation and application to field collected snow samples. Atmospheric Measurement Techniques. Retrieved May 13,
Delesky, E. A., Thomas, P. E., Charrier, M., Cameron, J. C., & Srubar 3rd, W. V. (2021). Effect of pH on the activity of ice-binding protein from Marinomonas primoryensis. PubMed. Retrieved Apr 27, 2021, from https://pubmed.ncbi.nlm.nih.gov/33090301/
Dolev, M. B., Bernheim, R., Guo, S., Davies, P. L., & Braslavsky, I. (2016, Aug 1). Putting life on ice: bacteria that bind to frozen water. The Royal Society Publishing. Retrieved Apr 28, 2021, from https://doi.org/10.1098/rsif.2016.0210
Duilio, A., Tutino, M. L., & Gennaro Marino, G. (2004). Recombinant protein production in Antarctic Gram-negative bacteria. PubMed. Retrieved Apr 27, 2021, from https://pubmed.ncbi.nlm.nih.gov/15269427/
Hill, R., Saetnan, E. R., Scullion, J., Jones, D. G., Ostle, N., & Edwards, A. (2015, Sep 16). Temporal and spatial influences incur reconfiguration of Arctic heathland soil bacterial community structure. PubMed. Retrieved Apr 28, 2021, from https://pubmed.ncbi.nlm.nih.gov/26259508/
Kassmannhuber, J., Mauri, S., Rauscher, M., Brait, N., Schöner, L., Witte, A., Weidner, T., & Lubitz, W. (2020). Freezing from the inside: Ice nucleation in Escherichia coli and Escherichia coli ghosts by inner membrane bound ice nucleation protein inaZ. PubMed. Retrieved Apr 27, 2021, from https://pubmed.ncbi.nlm.nih.gov/32429672/
Kassmannhuber, J., Rauscher, M., Schöner, L., Witte, A., & Lubitz, W. (2017). Functional display of ice nucleation protein inaZ on the surface of bacterial ghosts. NCBI. Retrieved Apr 27, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639866/
Lindow, S. E., Arny, D. C., & Upper, C. D. (1982). Bacterial Ice Nucleation: A Factor in Frost Injury to Plants. NCBI. Retrieved Apr 27, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1065830/?page=1
NASA’s Earth Science News Team. (2020, Nov 5). The Anatomy of Glacial Ice Loss. NASA Global Climate Change. Retrieved Apr 28, 2021, from https://climate.nasa.gov/news/3038/the-anatomy-of-glacial-ice-loss/
Parrilli, E. (2008, Feburary 7). Development of an improved Pseudoalteromonas haloplanktis TAC125 strain for recombinant protein secretion at low temperature. SpringerLink. Retrieved April 30, 2021, from https://link.springer.com/article/10.1186/1475-2859-7-2
Romanenko, L. A., Uchino, M., Mikhailov, V. V., Zhukova, N. V., & Uchimura, T. (2003). Marinomonas primoryensis sp. nov., a novel psychrophile isolated from coastal sea-ice in the Sea of Japan. PubMed. Retrieved Apr 27, 2021, from https://pubmed.ncbi.nlm.nih.gov/12807208/
Sannino, F., Parrilli, E., Apuzzo, G. A., Pascale, D. d., Tedesco, P., Maida, I., Perrin, E., Fondi, M., Fani, R., Marino, G., & Tutino, M. L. (2016, Oct 29). Pseudoalteromonas haloplanktis produces methylamine, a volatile compound active against Burkholderia cepacia complex strains. PubMed. Retrieved Apr 28, 2021, from https://pubmed.ncbi.nlm.nih.gov/27989956/
Sherman, D. G., & Lindow, S. E. (1993, Nov 1). Bacterial ice nucleation: significance and molecular basis. The FASEB Journal, 7(14), 1338-1343. Retrieved Apr 28, 2021, from https://doi.org/10.1096/fasebj.7.14.8224607
Tao, X., Feng, J., Yang, Y., Wang, G., Tian, R., Fan, F., Ning, D., Bates, C. T., Hale, L., Yuan, M. M., Wu, L., Gao, Q., Lei, J., Schuur, E. A. G., Yu, J., Bracho, R., Luo, Y., Konstantinidis, K. T. T., Johnston, E. R., … Zhou, J. (2020, Jun 5). Winter warming in Alaska accelerates lignin decomposition contributed by Proteobacteria. NCBI. Retrieved Apr 28, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275452/
Tutino, M. L., Parrilli, E., Giaquinto, L., Duilio, A., Sannia, G., Feller, G., & Marino, G. (2002). Secretion of α-Amylase from Pseudoalteromonas haloplanktis TAB23: Two Different Pathways in Different Hosts. NCBI. Retrieved Apr 27, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC139624/
Vance, T. D.R., Graham, L. A., & Davies, P. L. (2018, Apr). An ice‐binding and tandem beta‐sandwich domain‐containing protein in Shewanella frigidimarina is a potential new type of ice adhesin. The FEBS Journal. Retrieved Apr 27, 2021, from https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.14424
Xin, X. F., Kvitko, B., & He, S. Y. (2018). Pseudomonas syringae: what it takes to be a pathogen. NCBI. Retrieved Apr 27, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972017/
Zhang, L., & Xie, G. (2007, Jan 1). Diversity and distribution of Burkholderia cepacia complex in the rhizosphere of rice and maize. FEMS Microbiology Letters, 266(2), 231-235. Oxford Academic. Retrieved Apr 28, 2021, from https://doi.org/10.1111/j.1574-6968.2006.00530.x