Team:UNIK Copenhagen/Achievements

Achievements of the Red Lab


Summary:


  1. Assessment of S. elongatus and Bacillus subtilis survival after exposure to Mars-like conditions.
  2. Adaption and acclimation of S. elongatus and B. subtilis to Martian environment.

To know exactly how this is achieved, see Red Lab Overview and Experimental Concept.

GROWTH EXPERIMENTS – Validating the acclimation of bacteria based on the assessed
knowledge of the bacterial response to exposed Martian-like conditions

Question How well did the bacteria, S. elongatus and B. Subtilis, survive the different simulations of Martian environment?

Experiment The experiment at JMMC (Jens Martin Mars Chamber), with independent exposure to UV, pressure, temperature and microgravity (Random Positioning Machine) have been assessed. The bacteria were exposed for different duration times shown in the table below. The exposure times in the pressure and temperature experiments were combined with several pressure and temperature values respectively. Thus, the cells were immediately analyzed with OD (Optical Density) and microscopy to observe their response on change in growth rate. The B. subtilis were grown on LB media whereas S. elongatus were grown on BG11 media. The experimental assessment were designed for plated bacteria thus investigated in liquid form for the OD.

NOTE: not all microscopy images and OD measurements for the different experimental values are provided here due to simplicity. Therefore, microscopy pictures and diagrams are chosen to be shown from different experimental observations to illustrate the broad assessment.


Results Generally, the S. elongatus and B. subtilis have survived the Martian conditions upon exposure even on long durations, which is demonstrated on the pictures and growth diagrams shown below. The overall results extracted from the experiments also indicates the decrease in growth rate in an amount dependent on exposure durations referring to drastic drop for the longest durations and slighter fall for shorter exposure times. In the case of UV exposures, Martian UV spectra and single wavelength 293 nm, demonstrated that intensities matter significantly. The intensity of Martian UV spectra is higher than that of single wavelength despite the fact that it is a short wavelength, the higher power has caused the decreased tolerance to duration of exposure. Additionally, this fact of power appears in the graphs and microscopy pictures of pressure exposures. The exposures of low pressures in comparison to higher pressures, with the same duration time has slower growth rate, indicating the influence of strength of the exposing parameter. Visually, the transition of size can be seen on the microscopy pictures in fact for low pressure values the bacteria reshapes morphologically and goes from elongated shapes to circle-alike shapes. Despite the fact that the bacteria changes shapes, size and demonstrates decreased growth rates, they still survive Martian-like conditions.

Parameters

Exposure time for each parameter value

Pressure (kPa) : 0.5, 0.7, 0.9, 1.1

0 min, 6h, 3 days, 7 days

UV radiation (293nm)

0 min, 1 min, 2 min, 4 min, 8 min, 10 min, 30 min

Simulated Martian radiation (245-340nm)

0 min, 1 min, 5 min, 10 min, 15 min, 30 min

Microgravity : 0.4g

0 min, 6h, 4 days, 7 days

Temperature (°C) : +4, -20, -80

1h, 5h, 3 days, 7 days


Wild type bacteria

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Figure 1: Wild type Cyanobacteria S.elongatus (left) and Bacillus subtilis (right).

Pressure

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Figure 2: Bacillus subtilis subjected for 3 days to 0.5 kPa (left) and 0.7 kPa (right).

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Figure 3: Bacillus subtilis subjected for 3 days to 0.9 kPa (left) and 1.1 kPa (right).

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Figure 4: OD measurements diagram of Bacillus subtilis subjected for 6h to simulated Martian pressure.

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Figure 5: Cyanobacteria S.elongatus subjected for 0 min to 0.5 kPa (left) and 0.7 kPa (right).

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Figure 6: Cyanobacteria S.elongatus subjected for 0m to 0.9 kPa (left) and 1.1 kPa (right).

UV radiation

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Figure 7: Bacillus subtilis subjected to UV radiation (293nm) for 5 min (left) and 10 min (right).

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Figure 8: OD measurements diagram of Bacillus subtilis subjected to UV radiation (293nm).

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Figure 9: Cyanobacteria S.elongatus subjected to simulated Martian UV radiation for 5 min (left) and 10 min (right).

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Figure 10: Cyanobacteria S.elongatus subjected to simulated Martian UV radiation for 15 min (left) and 30 min (right).

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Figure 11: OD measurements diagram of the Bacillus subtilis subjected to simulated Martian UV radiation. .

Microgravity

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Figure 12: Bacillus subtilis unsubjected to microgravity (left) and subjected to microgravity for one week (right). Copyright iGEM Team Leiden 2016.

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Figure 13: OD measurements diagram of Bacillus subtilis subjected to microgravity. Copyright iGEM Team Leiden 2016.

Temperature

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Figure 14: Bacillus subtilis subjected for 5h to -80 °C (left) and -20 °C (right).

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Figure 15: Bacillus subtilis subjected for 3days to -80 °C (left) and -20 °C (right).

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Figure 16: Bacillus subtilis subjected for 1h to -80 °C (left) and -20 °C (right).

Question Was it possible to simulate all Martian parameters for combo-exposure experiment?

Experiment The bacteria were exposed to the UV at the specific duration times and immediately put in the little glass chamber and subsequently in JMMC for further pressure exposure. As both bacteria have shown good results in growth rate for pressure exposures on different durations, an extensive exposure have been carried out. In order to be able to do experiments in a row, the plates needed to be exposed to UV at first then exposed further and pulled out to stay it the created environment in the glass chamber for the chosen duration. Thus, the UV and pressure were not done at the same instant.


Results The growth rate of B. Subtilis has decreased dramatically in this combo-experiment, as expected. From previous experiments on Martian UV spectra assessment it was shown that the growth rates were affected notably therefore the results are consistent with the predicted behavior. The different exposure durations were chosen in order to predict and create a basis for coming experiments on adaptation and acclimation setup. The setup of 0.5 kPa, 30 mins of UV and 1H exposure has shown excellent performance in comparison to the other curves despite the fact that it is the lowest pressure value, and longest UV exposure duration, it is among quickest growing population.

Combo experiments

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Figure 17: Bacillus subtilis subjected for 1h to 0.5 kPa and for 15 min (left) and 30 min (right) to UV radiation.

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Figure 18: Bacillus subtilis subjected for 1h to 0.8 kPa and for 15 min (left) and 30 min (right) to simulated Martian UV radiation.

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Figure 19: Bacillus subtilis subjected for 6h to 0.5 kPa and for 30 min to simulated Martian UV radiation (left). OD measurements diagram of the Bacillus subtilis subjected to simulated Martian UV radiation and pressure (right).

Question How well did the S.elongatus and B. subtilis adapt to the Martian conditions?

Experiment Due to the breakage of JMMC and lack of time for reparation, the adaption experiments are carried out in another facility at the University of Copenhagen, Department of Plant and Environmental Science. At first the plates are exposed to UV single wavelength of 293 nm and subsequently exposed to pressure of 0,8 kPa for a duration of approximately seven minutes. The replating of exposed colonies will take place at their stationary phase, then incubated overnight in order to grow for another round of exposure with the same premises and values as applied in the first round. Growth rate assessments and physical changes of bacteria will be carried out by the means of OD and microscopy at an instant of each cycle. This procedure will demonstrate their ability of adaption and acclimation of the bacteria to Martian conditions.


Results The graphs and microscopy images of the adaption and acclimation results will be present at the presentation and included in the poster for the Giant Jamboree in Boston.

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Figure 20: Vacuum facility as a substitution to the JMMC (left). First and second round of bacterial adaption procedure (right).

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