Ice Nucleation

Pseudomonas syringae is well known for its ice-nucleating properties. This ability to freeze water at higher than normal temperatures helps the bacteria get on the ground from the clouds – by inducing precipitation. Humans have been trying to induce precipitation since the 1940s to this day using much the same mechanism – except the ice nucleating agent in this case is the chemical Silver Iodide, which when sprayed into clouds freezes cloud droplets which then fall to the ground. Pseudomonas are a better ice nucleator, and are commonly used in production of artificial snow or ice rinks, but not – as far as I know – sprayed into clouds. Other bacteria have an ability to do the opposite – act as ‘antifreeze’ by lowering the freezing point of water. The genes for these two proteins were engineered into one bacteria by an iGem team in 2011. The team’s project also proposed to engineer a genetic switch to turn on either of the genes by some outside input. We will be building on their project to create a switch which would introduce even more control over bacteria’s time of transit in the atmosphere.

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The plasmid with the inaZ gene (used by the iGem team) was inserted into e. coli bacteria, which is safe to use in a lab, and saved for future use. Plasmids are circular pieces of DNA that can be inserted into the bacteria. In higher organisms the DNA inserted into a cell would be integrated into its host genome. But bacteria have one circular chromosome and the plasmids we’re introducing are not integrated into its genome but float freely in the cell. Our synthesized DNA will also be inserted as a plasmid into the bacteria.

We did the ice nucleation test at Genspace (with the help of Will, Julie and Blacki). We prepared a negative control of e. coli bacteria without the gene, and followed iGem’s protocol:
An overnight preculture was inoculated at an initial OD of 0.1 in 15mL medium, and grown for 5 hours at 37°C. Cells were spun down and resuspended in 1 mL filtered water, after which they were washed twice with 1mL filtered water. The ODs of the resulting cell suspensions were determined and all cells were diluted to the same OD of 10. In the meanwhile, a cooling bath was prepared to a temperature around -10°C, and clean, plastic tubes filled with 5mL filtered water were put in the chilled bath to create supercooled water. The same amount of INP-expressing or control cells were added to the 5mL supercooled water and checked for ice nucleating activity.

Addition of bacteria expressing INP to supercooled water induced ice crystallization earlier than with addition of bacteria without the INP gene. We discovered that the difference between the two is fairly narrow and getting a nice result of water turning into ice as if by magic requires precise control of the temperature of the water, and it took us a few tries to try to demonstrate it. For example in the first try I took the tubes out of the cooling bath a few minutes before adding the bacteria, and the water didn’t freeze in either sample. But after putting the samples in the cooling bath again, one with INP froze after a few minutes while the control remained liquid.

iceNucleation from amateurhuman on Vimeo.

iceNucleation control from amateurhuman on Vimeo.

Here’s our supercooled water turned into ice with the help of the bacteria.

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And the plasmid map of the plasmid containing inaZ gene. It is prepared using synthetic biology tools that attempt to standardize biotechnology techniques, approaching biology with an engineering mindset.

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Pseudomonas syringae produces a protein on its surface which promotes water molecules to crystallize into ice at temperatures higher than normal. The bacteria is associated with all the environments in hydrological cycle, including being well adapted to the atmosphere. Its role in controlling precipitation patterns and potentially in energy budget, physiochemical processes of the atmosphere and hydrological cycle are just beginning to be mapped out. But ice nucleation has been linked to inducing precipitation since scientists at GE (including Bernard Vonnegut, brother of novelist Kurt Vonnegut) used silver iodide in 1946 to successfully precipitate a cloud – literally creating a hole in it by freezing those droplets to let them fall down to the ground. GM at the time was tasked with research in weather modification. Today silver iodide is a standard agent used in cloud seeding: “approximately 50,000 kg are used for cloud seeding annually, each seeding experiment consuming 10–50 grams.” Kurt Vonnegut, who also worked at GM at the time – in the PR department, was inspired by these experiments to predicate his next novel, Cat’s Cradle, on an invention of a substance (Ice-Nine) which could freeze water at room temperature, which led to the Earth freezing over.

GE research scientists Irving Langmuir, Bernard Vonnegut, and Vincent Schaefer are seeding a snow cloud:

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The ice nucleating gene has been of interest to scientists for a long time, partly because it is responsible for early frost causing substantial damages in agriculture. In 1987 Pseudomonas syringae with the ice-nucleating gene snipped out through genetic engineering was tested on a field of potatoes – a very first field trial of any GMO. The trial sparked a long-lasting debate on safety and remifications of genetic modification.

Berkeley plant scientists under direction of Steven Lindow spraying a field of potatoes with ice-minus, a genetically engineered version of Pseudomonas syringae that prevents frost, in 1987.

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As a member of atmospheric microbiome, Pseudomonas syringae deserves special attention. It thrives in the clouds, with high resistance to UV, cold and salinity, and with an ability to utilize air pollutants as nutrients. It is found in all habitats associated with the water cycle. Its size, buoyant density and surface properties determine its high capacity to remain aloft in the air. Studies have shown that the bacteria not only passively travel in the clouds, but are also metabolically active there and might effect the physicochemical properties of the atmosphere. In particular, P. syringae’s ability to induce clouds to form and precipitate might influence solar radiation balance and hydrologic cycle.

Plants are the most important source of microorganisms in outdoor air. The studies of plant-bacteria interactions developed into what became a new field of aerobioogy in the 1900, when American farmers started planting a new hardy crop: Crimean variety of winter wheat released by scientists from the US Department of Agriculture. The wheat monoculture spread from Mexico to Canada creating what was termed the ‘grain belt.’ This plant monoculture was devastated by epidemics of stem rust caused by a pathogenic microorganism which – as it turned out – travelled through the air for many miles. To control the epidemic, a fleet of planes was deployed sampling the atmosphere at all altitudes to map out the organisms’ presence in the air. These samplings demonstrated that microorganisms are present and common at altitudes of clouds and beyond.

Covering the landscape with particular vegetation thus creates conditions for proliferation of microorganisms that favor it, which in turn might have an effect on the processes in the atmosphere and even subsequently affect the climate.

To support our cloud bacteria we are preparing a plant that would nurture and amplify it when it is on the earth’s surface. The plant will be based on a red clover, which is already present in most temperate environments around the globe, thrives in most soils and, as nitrogen fixer, is already mixed in with many crop plants.