Transcript Mission 7, Flight Experiment

Mission 7
How is the Growth of the Bacteria Rhizobium
radiobacter Affected by Microgravity?
Montachusett Regional Vocational Technical School
Fitchburg, Massachusetts
Team Members
Marina Good: Principal Investigator
Jezrielle Bruno: Collaborator
Teacher Facilitator - Paula deDiego,
Monty Tech
The Proposal
The experiment was focused on how the growth of the
bacterium, Rhizobium radiobacter, and by extension, the
production of endotoxins, could be affected by microgravity,
as opposed to its observed growth and endotoxin production
on Earth.
Our Predictions
Freeze-dried Rhizobium radiobacter can be successfully
activated and grown in microgravity.
More bacterial growth and higher endotoxin production will
occur as a result of microgravity.
Our Packaging and Interactions
Packaging:
- Volume 1: Freeze-dried bacteria - 1 ampoule of
R. radiobacter
- Volume 2: Growth Media - Filled to capacity
with Luria Broth (LB)
- Volume 3: Growth Inhibitor - 1 mL of 0.3 M
Acetosyringone
Interactions:
U-5 [May 7th, 2016] - green clamp opened
(activated experiment), inverted for 30 sec,
mixing bacteria and growth media.
U-2 [May 9th, 2016] - blue clamp opened
(deactivated experiment), inverted tube for 30
sec, mixing growth inhibitor.
Analyses Performed
Optical Density Test
Supernatant
Microscopy Analysis
Mass Pellet Test
Endotoxin LAL Biological Assay
Extraction from cell membrane
Supernate
Results for Optical Density ~ Supernatant
Optical Densities
Procedure
1 mL of each sample was taken, serially
diluted 6 times (⅙ dilution), and analyzed
at 600 nm, each cuvette containing 900
microliters of liquid.
*Note: The serial dilution was made in triplicate.
Results were recorded at dilution (10⁻¹)
with 900 microliters of liquid in the
cuvette.
Ground Truth
Tubes
OD’s
Flown Tubes
OD’s
GA
0.384
SA
0.372
GB
0.381
SB
0.392
GC
0.394
SC
0.406
Average OD
of Ground
0.386
Average OD
of Flown
0.39
This means that, based on these observations, there were virtually the same amount of cells (and growth)
present in both samples, although the Flown Samples had a slightly higher average OD (0.39).
Microscopy of R. radiobacter
Procedure:
Both samples were streaked on slides, stained red (gram negative), and viewed underneath a
microscope.
Observations:
The bacterial colonies of the Ground Truth were more compact, while those of the Flown
Sample appeared slightly more spread out within their groupings.
The cells of both samples were rod-shaped, as expected of a bacillus bacteria, but the cells of
the Flown Sample appeared slightly rounded, while the cells of the Ground Truth were more
distinctly rod-shaped.
Results for Mass Pellet
7 mL of each sample were
spun in 14 microcentrifuge
tubes at 14,000 RPM for 15
minutes.
The supernatant was drawn
off, and the masses of each of
the remaining pellets of cell
debris were taken.
Key:
G - Ground Truth [Ground]
S - Flown Sample [Space]
Tube(1 mL
per tube)
Empty Tube
(g)
Tube w/
Pellet (g)
Pellet Mass
(g)
G1
0.9849 g
0.9967 g
0.0118 g
G2
0.9883 g
1.0006 g
0.0123 g
G3
1.0168 g
1.0285 g
0.0117 g
G4
0.9947 g
1.0062 g
0.0115 g
G5
1.0112 g
1.0502 g
0.039 g
S1
1.0044 g
1.0149 g
0.0105 g
S2
0.9852 g
0.9975 g
0.0123 g
S3
1.0060 g
1.0194 g
0.0134 g
S4
1.0014 g
1.0127 g
0.0113 g
S5
0.9992 g
1.0139 g
0.0147 g
Average Mass
(g)
0.01726 g
0.01244 g
Biological Endotoxin Assay for R. radiobacter
The presence of endotoxins was confirmed after appropriate extraction
from, and preparation of, both samples was complete, as well as an Optical
Density Test, run at 545 nm.
The high detection test was used, meaning that samples with added
chemicals were incubated for 7 minutes (Time 2 [T2]) as indicated by the
manufacturer. It was assumed that because of the cloudiness of the
samples, there were large amounts of bacteria, resulting in high levels of
endotoxins, requiring the high detection test, as opposed to the more
sensitive low detection test (Time 1[T1]), used for lower concentrations of
endotoxins.
*Note: The Limulus Amebocyte Lysate (LAL) was slightly cloudy, so it was swirled for more than 30 seconds.
**Note: The endotoxin was fully dissolved.
Results for Optical Density ~ Endotoxin Assay
Extracted Endotoxins
The high detection test was used, meaning that
samples (with added chemicals) were
incubated for 7 minutes (Time 2 [T2]) as
indicated by the manufacturer.
Samples were run at 545 nm, with 1 mL of
liquid in each well.
Samples were diluted (1 x 10⁻¹)
100 microliters of sample
(supernatant) in 900 microliters of
water (H₂O)
Optical Density Data Table ~ Endotoxin Assay
Standards:
Concentration Avg. Optical Density Minus Blank
Blank
0.113 (Minus Blank Value)
1 EU/mL 1.084666667
0.9716666667
0.5 EU/mL
0.765
0.652
0.25 EU/mL
0.643
0.5303
0.1 EU/mL
0.42
0.307
Sample
Average Optical
Density
Subtracted Blank
OD (0.113)
Flown
Supernatant
(Diluted [10⁻¹])
1.266333333
1.153333333
Flown Sample
(Undiluted [10⁰] Extracted)
1.212
1.099
Ground Truth
Supernatant
(Diluted [10⁻¹])
1.235666667
1.122666667
Ground Truth
(Undiluted [10⁰] Extracted)
1.169
1.056
*Note: EU/mL means endotoxin unit per milliliter.
Results for Optical Density ~ Endotoxin Assay
Sample
Endotoxin
Concentration
(EU/mL)
Ground Truth
Sample
(Undiluted [10⁰] Extracted)
0.275 EU/mL
Ground Truth
Supernatant
(Diluted [10⁻¹])
0.293 EU/mL
Flown Sample
(Undiluted [10⁰] Extracted)
0.287 EU/mL
Flown Supernatant
(Diluted [10⁻¹])
0.302 EU/mL
*Note: EU/mL stands for Endotoxin Unit per milliliter.
Benefit of Knowledge Gained
The Optical Density of the Flown Supernatant was slightly higher than that of the Ground Truth Supernatant,
but not remarkably so.
The bacterial colonies of the Flown Sample were not as compactly grouped as those of the Ground Truth.
The bacteria of the Flown Sample appeared slightly rounded in shape, while the bacteria of the Ground Truth
were more distinctly rod-shaped.
The mass of the Flown Sample pellet was slightly lower than that of the Ground Truth pellet, but not
remarkably so.
The endotoxin concentration of the Flown Sample (of both the diluted supernatant and the undiluted sample
extracted from the membrane) was higher than that of the Ground Truth (in both the diluted supernatant
and undiluted sample extracted from the membrane).
Future Questions
How is the structure and appearance of the cell membrane of Rhizobium radiobacter affected
by microgravity?
If samples of Rhizobium radiobacter were grown in space, returned to Earth, and continued to
be grown, would the endotoxin production differ compared to a sample grown continuously
on Earth?
Would the genetic modification in plants, caused by Rhizobium radiobacter, be different if the
bacteria was grown in space?
As a commonly used vector in genetic modification and engineering in plants, would
Rhizobium radiobacter grown in space be more efficient, less efficient, or even alter the nature
of the introduction of a foreign gene?
Mentors ~ Thank you!
Dr. Kristen DeAngelis University of Massachusetts
Amherst Microbiology lab
Doctoral Candidate Gina Chaput University of
Massachusett Amherst Microbiology lab
Sponsors
North Central Community Foundation -Debbie & Phillips
Richards
Questions?