Team:Stanford-Brown/VenusLife/Chamber

Future Directions

Amatya-Jackson Ltd.

ISS Bacterial Aerosol Experiment Proposal

Intro:

The creation of a reliable cell cycle dependent promoter has opened the door for powerful, new astrobiological experimentation techniques. We wish to apply this tool to study bacterial life in aerosol under null gravity and varying atmospheric conditions, using either bioluminescent or fluorescent signal reporters. Specifically we aim to achieve two goals: First, to measure bacterial growth in aerosolized lab media droplets, and second, to measure bacterial growth in Venusian atmospheric conditions with the potential for directed evolution based development of “Venus-grade” bacteria.

Atmospheric Chamber Design:

This sort of experimentation requires the creation of an atmospheric chamber to house the bacteria and test their viability. Because we want our bacteria constrained to the aerosol, the inner surfaces of the box should be constructed with antibacterial material. Additionally, the box should be outfitted with an input and output tube and valve apparatus to easily add chemical compounds, inject the aerosolized bacteria, and draw samples from the chamber. The box should also have a temperature and pressure regulation system. Finally, the chamber must have a viewing scope and detection apparatus corresponding to the reporter connected to the nrd promoter. Fluorescence requires a specific excitation wavelength and light filters on the camera. For bioluminescent detection, the chamber could be equipped with a charge-coupled-device (CCD) camera, or a photon-counting video camera (1).

Potential Experiment 1:

Goal: Determine if standard E. coli can divide in aerosolized media at standard Earth conditions (temperature and pressure, but with no gravity) Bacteria: E. coli transformed with the cell cycle dependent promoted reporter Media: Try various medias of different nutrient levels to determine the limits of survivability Procedure: 1. Inject bacteria in aerosol solution using atomizer through input valve. Bacteria are suspended in chamber because of 0 gravity. 2. Set chamber to Earthlike standard conditions, and cover box with dark material to eliminate background light 3. Set camera to record chamber. Look for signal expression.

Experiment 2:

Goal: Determine whether other standard E. coli or other living or synthetic organisms can survive in aerosol under Venus-like conditions Bacteria: Standard E. coli, The Hell Cell, other synthetic organisms Media: Venusian air Procedure: 1. Inject organism in aerosol solution using atomizer through input valve. Organisms are suspended in chamber because of 0 gravity. 2. Set chamber to Venusian atmospheric conditions in 50-60km cloud layer, and cover box with dark material to eliminate background light Venusian atmospheric conditions: -Temperature: between -10 and 77 degrees C -Pressure: 1 atm -pH: 0 -Composition: 96% carbon dioxide, 3% nitrogen, and 0.003% water vapor (2). Small quantities for other gases can be found in source (3). 3. Set camera to record chamber. Detect signal.

Conclusion:

The nrd promoter construct could be useful in astrobiological experiments because it allows for detection of cell division without having access to or interfering with the cells themselves. The tunability of the signal can be paired with existing detection techniques as well, such as bioluminescence and fluorescence.

References:

1. Sternberg, Claus, Leo Eberl, Lars Kongsbak Poulsen, and Søren Molin. "Detection of Bioluminescence from Individual Bacterial Cells: A Comparison of Two Different Low-light Imaging Systems." Journal of Bioluminescence and Chemiluminescence 12.1 (1997): 7-13. Print. 2. Landis, Geoffrey A. “Astrobiology: The Case for Venus.” National Aeronautics and Space Administration. 2003. 3. http://en.wikipedia.org/wiki/Atmosphere_of_Venus#cite_note-Institutdemecanique-9

The Millikan Apparatus

In order to put our newly engineered organisms to the test in an aerosolized environment, the team aimed to modify a Millikan Oil Drop Apparatus into a functioning suspension chamber. Designed in 1909, the chamber was originally used by Robert Millikan and Harvey Fletcher to measure the elementary electric charge. The Apparatus is designed around a parallel pair of horizontal metal plates. Applying a potential difference across these plates creates a uniform electric field in the space between them, and thus charged droplets sprayed finely into the chamber can be made to rise and fall by altering the applied voltage. Instead of oil, we aimed to suspend a medium solution with our engineered biosensing bacteria in order to visualize whether these microorganisms were thriving in an aerosolized environment.

Unfortunately, we encountered a few engineering issues while preparing for this experiment:

• The engineering of the Millikan Apparatus viewing scope to excite and detect fluorescence was impractical and challenging.
• Control tests on evaporation in the chamber indicated that droplets would evaporate in under 10 minutes; a timescale too rapid for studying cell replication.
• Inconclusive evidence on whether or not the medium we were planning on using (SOC) could support bacteria on the order of hours and emit minimal to no autofluorescence.
• Insufficient time and resources to perform the necessary (and drastic) modifications to the Apparatus that would lead to successful experiments.

However, we successfully created cell-growth dependent promoters that function as remote biosensors; while our efforts in mechanical engineering were not so fruitful, the synthetic biology component was successful! In addition, our modeling component of this project provides insight and suggests that bacteria could feasibly inhabit the 50-60km layer of the Venusian atmosphere.

We would like to thank Diana Gentry for helping us with the mechanical engineering of the suspension chamber!

The Venus Life team presents the suspension chamber