Investigation of cosmic ray mutations in bacteria

Research Group: Sarah Burshek, Kasey Gladson, and Ashlyn Hill

Launch: Whitworth Spring 2022

Project summary goes here...

Near Space
Near space is between 65,000 feet and 325,000 feet above sea level, and it consists of multiple layers. The first layer in the Earth's atmosphere is the troposphere; this is the lowest layer extending from Earth's surface to an average height of about 33,000 feet above sea level. The troposphere is the densest of the layers and includes the most weather. The second layer is the stratosphere, starting at about 33,000 feet above sea level and extending to 164,000 feet. The stratosphere is also where the ozone layer resides. The third layer of the atmosphere is the mesosphere; it roughly ranges from 164,000 feet above sea level to 275,000 feet. The fourth of the five relevant layers is the thermosphere, which starts at about 275,000 feet above sea level and ends at the last layer, the exosphere (nearly 2 million feet above sea level). The fourth layer, the thermosphere, absorbs the brunt of cosmic radiation rays. In the first troposphere layer, the ozone is the last line of defense, and it scatters and absorbs what is left of the cosmic radiation rays; this makes it possible for Earth to be habitable.

Cosmic Rays
Cosmic Rays are fragments of atoms that fall to Earth from space; Cosmic radiation can be found in each atmospheric layer. The large majority of these rays do not make it to the Earth's surface due to the collisions with the atoms in the upper atmosphere. These collisions result in neutral pions, which quickly decay into high-energy gamma radiation. About 90% of these rays are hydrogen particles. They can range from high to low power, and their origin is not yet known. However, It is predicted that giant rays result from supernovas, and some smaller rays are released from the sun called solar flares. In 1912 Victor Hess flew a high altitude balloon 5,300 meters above sea level and found ionizing radiation three times higher than that on Earth's surface. The amount of cosmic rays observed in our atmosphere is dependent upon solar winds, Earth's magnetic field, and the energy of the individual rays.

Bacteria
Bacteria take at least 15 minutes for new generation growth and will grow between temperatures of 20 and 37℃. Radiation affects cell size by decreasing cell size by surface area, and an increase in cell envelope thickness. Bacteria are known to have very quick life cycles and grow extremely quickly. Escherichia coli is one of the most common types of bacteria that have been experimented on and researched for many years for its fast growth and easy genetic manipulation. Since the 1940s, E. coli has been a model organism because of its quick growth, previously mentioned, and is easy to manipulate.

Mechanical Design


Inside the pod was a wooden and cardboard stabilizer and LocknLock container. The wooden and cardboard stabilizer was to keep the box steady and prevent things from moving around. Inside the LocknLock container were two D batteries, each in their own container, two test tubes of liquid bacteria, and an in-flight computer with heaters, transistors, and temperature sensors attached. The two D batteries were powering the heaters to keep the LocknLock container between 21-24℃ so the bacteria don’t overheat nor freeze to death. The ideal bacterial growth range is 17-degrees (20-37℃), and in this case the range was 3-degrees and on the lower end of ideal bacterial to keep the batteries from overworking and being used-up on the ground. This saves battery by not having the heaters heat up to a higher heat. The point of this system was to keep ozone out and to not overheat or freeze the bacteria. The ozone was kept out by the airtight container. The heating system kept the bacteria at a surviving temperature.

The dimensions of the pod were 7.37in x 5.91 in x 4.29in. The dimensions of the container were 6.1in x 6.1in x 3.7in. Below is the link to where the container was purchased.

https://www.amazon.com/LOCK-Purely-Better-Container-Storage/dp/B084114H2M/ref=asc_df_B084114H2M/?tag=hyprod-20&linkCode=df0&hvadid=416675160954&hvpos=&hvnetw=g&hvrand=8006807538888397549&hvpone=&hvptwo=&hvqmt=&hvdev=c&hvdvcmdl=&hvlocint=&hvlocphy=9033791&hvtargid=pla-908931088759&psc=1&tag=&ref=&adgrpid=97671773727&hvpone=&hvptwo=&hvadid=416675160954&hvpos=&hvnetw=g&hvrand=8006807538888397549&hvqmt=&hvdev=c&hvdvcmdl=&hvlocint=&hvlocphy=9033791&hvtargid=pla-908931088759

Heating system
The heating system consists of three heaters attached to three transistors which were powered by two D-batteries. The heaters were attached to a wooden pole that fit diagonally across the LocknLock container. The heaters were 7.2 ohm resistors while the transistors were FQP30N06L MOSFET transistors. These were connected together, and connected to the two in-series D-batteries.

Batteries
The batteries were in a series. Since the two 1.5 V batteries were in series with each other, that made the batteries a total of 3 V together. The total power would be the total voltage squared (9 V^2) over resistance of 7.2 ohms, which equals 1.25 V. To find how long the battery would last, 16,000 mA over the number of resistor (3) times current (.42 A). The batteries should last 12.7 hours.

Programming
Two temperature sensors were connected to the mbed of the in-flight computer. With the heaters and transistors connected to the in-flight computer, the code programmed the heaters to turn on or off depending on what the temperature sensors read. The heaters were programmed to turn on when the container was lower than 21℃ and to turn off at 24℃.

Transistors and Heaters
FQP30N06L MOSFET Transistors were used. The diagram describes how they connect and work. G = Gate, D = Drain, and S = Source. Gate was the terminal controls the conductivity between the Source and Drain terminals. Drain was a terminal though which charge carriers leave the channel. Source was the terminal through which charge carriers enter the channel. The heaters were 7.2 ohm resistors that were wired to a transistor and to the mbed. The connection to the mbed allowed them to be programmed to turn on off when needed.

April 15, 2022
The three tests on April 15, 2022 were the pre-validation and calibration. Test 1 had the temperature sensors and mbed inside the closed container for 10 minutes. Test 2 had the temperature sensors testing for 10 minutes outside the container with varying temperatures. Test 3 the temperature sensors tested for 15 minutes inside the closed container.

May 5, 2022
The six tests on May 5, 2022 were post-validation and calibration. The system includes the heaters, transistors, batteries, in-flight computer, and temperature sensors. These temperatures were from the minute it took to get to the freezer, put the system (potentially pod and/or container) in the freezer, run for 10 plus minutes and the minute walk-time back until system was turned off. Test 1 had the system inside the pod haphazardly put together with the battery on and in the LocknLock container. Test 2 had the system outside of the pod in an orderly fashion (like on the day of launch) with the battery on and in the container. Test 3 had the system orderly inside the pod with the battery on and in the container. Test 4 had the system orderly inside the pod with no batteries on and in the container. Test 5 had the system was orderly inside the pod with the batteries off and no container. Test 6 had the system orderly inside pod with the batteries on and no container. To show a close-up on the short-term temperature tests, Test 1 was taken off of the close-up graph. Test 2 and 3 had the battery on and were in the containers and both of those had increases in temperature before being put in the freezer whereas the other tests without the batteries on and/or outside of the container did not have that increase in temperature. Test 3 starts at a lower temperature since, Test 2 had just taken place and the container was still cold to the touch. Based on these results, the system did help. The pod and container are both necessary to keep bacteria warmer. The heaters did not help too much in this case so either more heaters and/or increase starting heat temperature and coded temperatures.

Data and Analysis
The data collected was temperature vs time. This raw data were from the two temperature sensors. On average, the temperature decreases 13.7°C. Take-off took place around 2000 seconds and the balloon popped around 6800 seconds. When the balloon popped, it turned off the in-flight computer, which was when the data stopped being collected.



The two experimental test tubes and control test tube were diluted from the test tube to a broth until experimental test tube 1, experimental test tube 2, and control were 0.081, 0.082, and 0.085 absorbance, respectively, in the VWR brand, double bean UV-Vis Spectrophotometer, Model UV6300PC. The necessary absorbance was between 0.080-0.090. Then all three were diluted again 100nL of broth 1 into broth 2. Then that was further diluted to another 100nL into broth 3. The broths were the same BD Dilfco nutrient broth. These dilutions took place so the differences between the colonies of bacteria were easily noticeable. The bacteria were then smeared on 3 Dilfco nutrient agar plates of each. Three for experimental test tube 1, three for experimental test tube 1, and three for the control test tube. They were plated and placed in an incubator April 29th evening. May 2nd the plates were stored in a fridge to stop bacterial growth to analyze in the evening of May 3rd.

There wasn’t much difference between the experimental bacteria and control. They all were fuzzy, white colonies, the largest and smallest diameters were about the same, and there were more smaller diameter colonies than large ones across all 9 plates. Largest diameters were 0.5cm for experimental bacteria and control. The bacteria looked the same on May 3, 2022 when they were observed in detail as they do in the photograph taken May 11, 2022.

Conclusion
The system did work by keeping out ozone with the air-tight container, and it did keep the bacteria warm enough that they survived and were able to grow. There were no phenotypic differences between the control test tube and the experimental test tubes once they were plated and able to grow enough to be seen. They were both fuzzy, white colonies with the largest being 0.5cm. The less than two-hour exposure to cosmic rays in near space did not have visible phenotypic impact on liquid E. coli.

Future groups could improve on increasing the temperature range to higher values in the code or start the container off at the higher end such as 37℃ to verify that the cold did not make a difference in the growth. The E. coli would have had slower colony growth rate in the cold, but since all the test tubes started between 0.080-0.090 absorbance and then had the same dilutions, they all had about the same colony amount starting on the plates. Testing if adding more heaters and transistors may increase the heat. Another thing would be to do more calibration and validation before take-off.