Bacterial Photography Overview
Background
Synthetic Biology is a field that focuses on designing and engineering new biological systems
with unnatural functions, with applications ranging from health to industry. The young field has
seen rapid growth over the last decade.
Bacterial photography was introduced early in the field by Levskaya et al. in “Engineering
Escherichia coli to see light,” Nature 2005: “We have designed a bacterial system that is
switched between different states by red light. The system consists of a synthetic sensor kinase
that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern
of light on to the bacteria produces a high-definition (about 100 megapixels per square inch),
two-dimensional chemical image. This spatial control of bacterial gene expression could be
used to 'print' complex biological materials, for example, and to investigate signaling pathways
through precise spatial and temporal control of their phosphorylation steps.”
Safety
The bacteria used in this lab are genetically modified Escherichia coli K12 (non-pathogenic,
BSL1) and need to be handled and disposed of following standard BSL1 classroom protocols. If
biohazard containers are available, dispose contaminated materials inside. Otherwise, soak
contaminated materials in 10% bleach for 20 minutes (1 hour for petri dishes). Wipe
tabletops with 10% bleach. Pour excess liquid down the drain, and double-bag materials to
dispose of in normal trash. Hands should be washed before leaving the laboratory.
Provided materials:
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Bacterial strain transport method:
*Note: We have 4 methods of pre-culturing bacteria, for testing. See the attached
sheet for more details and instructions.
o 2 14mL already pre-cultured tubes
o 2 glass vials of bacteria strain + 2 14mL culture tubes
Transparency paper
5mL tubes of photography media, containing growth media, agarose gel, antibiotics, and
supplemented tryptophan at appropriate concentrations
60mm Petri Dishes
Light chambers
Power supply (12V)
Black sharpie pen
Classroom supplied materials:
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10% bleach
Clear tape, scissors
pipettes capable of 0.1mL (100uL) volumes
150mL Beakers
30°C & 37°C incubator
Hot plates
(optional) Bunsen Burner
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Classroom Protocol
Storage
1. Store the 4mL vials of bacteria, 5mL tubes of photography media, and 14mL tubes of
growing media at room temperature.
Day 0: Bactograph Pre-Culture
Materials: 10% bleach, Bunsen burner, 4mL vial with bacteria, pipettes, 14mL culture tube with
growing media, 14mL culture tubes with pre-culture, 37°C incubator
1. Wipe down working space with 10% bleach. (optional) Light the Bunsen burner and
place near workspace. This will help create a sterile environment.
2. Follow protocol in appendix A based on method of pre-culture.
3. The culture should be a deep blue color after incubation; cultures that are not visibly blue
will NOT produce successful bacterial photographs.
4. Dispose of materials that have touched the bacteria following the given safety protocols.
5. Wash your hands with soap.
Day 1: Preparing the photograph
Materials: 10% bleach, Bunsen burner, hot plate, 5mL tube of photography media, 100mL
beaker, Day 0 Bactograph Pre-Culture in 14mL culture tubes, pipette, 60mm petri dish, sharpie,
transparency, clear tape, scissors, light chamber, 12V power supply, 30°C incubator
Melting the photography media (steps 2-3) takes at least 20min. The instructor may
choose to perform these steps prior to the class starting.
1. Wipe down the work space with 10% bleach. (optional) Light the Bunsen burner and
place near workspace. This will help create a sterile environment.
2. Add 50mL of water to the 100mL beaker, and place it on the hot plate. Turn on the
hot plate to at least 70°C.
3. Place the 5mL tubes of photography media inside the beaker. Allow the photography
media to liquify (at least 20min)
4. Using a sharpie, draw an image on the transparency with thick lines.
*Note: The image should be no larger than 50mm in diameter.
*Caution: The transparency paper is write-on only. Do not put through printer or copier.
5. Allow image to dry. Retrace the image 3 times to make the lines more opaque. Cut out
the image.
6. After the photography media liquefies, remove the tube from the beaker and shake well
to melt.
Complete steps 7-11 (below) as quickly as possible to prevent the photography media
from solidifying before it is spread out on the plate.
7. Remove Day 0 Bactograph pre-culture from 37°C incubator. Transfer 100uL (0.1mL) of
bacteria solution from the 14mL culture tubes to the 5mL tube of photography media.
o Note that the pre-culture must be blue/deep blue for a successful bactograph.
Pre-cultures that are not blue will not produce successful bactographs.
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8. Ensure that the cap of the 5mL photography media tube is tightly closed. Mix the
photography media by gently inverting (to prevent bubbles forming) 10-20 times.
9. Pour the photography media into a 60mm petri dish.
*Note: If the photography media solidifies before pouring, it can be reused. Re-prepare
following steps 2-6. New bacterial cells must be added.
10. Carefully rock the petri dish back and forth to evenly distribute the media across the
bottom all the way to the edges, and quickly remove any bubbles within the petri dish by
poking with a sterile pipette tip.
11. Close the petri dish and wait for the photography media to cool and solidify (at least
5min).
12. Turn the petri dish upside down (smaller side facing up). Tape the transparency image to
the face of the small side using minimal amounts of clear tape.
13. Put the light chambers in a 30°C incubator. Plug the 12V power source into light
chambers to turn on the LEDs. Check to make sure the lights are on.
14. Place the petri dish in a light chamber with the mask facing up. Make sure no outside
light enters the light chambers from the top, bottom, or sides.
15. Dispose of materials that have touched the bacteria following safety protocols.
16. Wipe down table tops with 10% bleach. Wash your hands with soap and water.
Day 2: Observing the Photograph
*Note: Bacteria must be in chambers for at least 24 hours! However, they can stay
incubated up to 72 hours, which may result in better photographs.
1. Remove petri dish from the light chamber, remove mask and look at the bacterial
photograph! *Note: Holding the petri dish up to a light may help to bring out the
image.
2. Dispose of materials that have touched the bacteria following the safety protocols
highlighted earlier.
a. Wipe down light chambers with 10% bleach
*Note: The bacterial photographs can be stored for several months at 4°C before they
need to be disposed of properly.
3. Wash your hands with soap and water.
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Appendix A: Day 0 Bactograph Pre-Culture Protocol
Introduction: We have had problems in the past with the bacterial pre-culture growing too slowly
in the absence of a shaking incubator. In an attempt to find a solution, we have included four
pre-culture methods with this kit. We ask that the group of participants be evenly divided
into groups to test these four methods.
Please follow the instructions below for your method:
Method 1: Pre-culture Already Grown
1. Locate the 14mL culture tube labeled ‘Pre-Culture Grown’. This tube has already been
inoculated with bacteria and put into a 37°C shaker in a lab for 48 hours before transit.
2. Place the tube into a 37°C incubator. No bacterial inoculation necessary.
Method 2: Pre-culture In-Transit
1. Locate the 14mL culture tube leveled ‘Pre-Culture In-Transit’. This tube was inoculated
with bacteria immediately before shipping and has been growing since.
2. Place the tube into a 37°C incubator. No bacterial inoculation necessary.
Method 3: Bacterial Strain Already Grown
1. Locate the 4mL glass vial labeled ‘Bacteria’ on top and ‘Grown’ on bottom. This bacterial
strain was stabbed into the agar 48 hours before shipping.
2. Locate the stab site in the 4mL vial of bacteria. Using a pipette, stab into the same area
and dig out a small amount of bacteria.
3. Place the pipette tip into a 14mL culture tube labeled ‘Growing Media’.
*Note: Do not accidentally use the 14mL culture tubes labeled ‘Pre-Culture’.
4. Pipette up and down to mix.
5. Cap the 14mL culture tube with the looser cap setting.
6. Place the tube into a 37°C incubator.
Method 4: Bacterial Strain In-Transit
1. Locate the 4mL glass vial labeled ‘Bacteria’ on top and ‘In Transit on bottom. This
bacterial strain was stabbed into the agar right before shipping.
2. Locate the stab site in the 4mL vial of bacteria. Using a pipette, stab into the same area
and dig out a small amount of bacteria.
3. Place the pipette tip into a 14mL culture tube labeled ‘Growing Media’.
*Note: Do not accidentally use the 14mL culture tubes labeled ‘Pre-Culture’.
4. Pipette up and down to mix.
5. Cap the 14mL culture tube with the looser cap setting.
6. Place the tube into a 37°C incubator.
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Appendix B: Kit Inventory
(18 people)
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1 4mL vial containing optogenetic bacterial strain grown in lab
1 4mL vial containing optogenetic bacterial strain grown in transit
1 14mL Bacteria culture tubes with pre-culture grown in lab
1 14mL Bacteria culture tubes with pre-culture grown in transit
2 14mL Bacteria culture tubes with 5mL growing media
 Growing media: LB, Tryptophan, Antibiotics (Ampicillin, Spectinomycin,
Chloramphenicol)
20 pieces of transparency paper
3 Black Sharpie markers
18 5mL tubes of photography media
 Photography media: LB Agar, Tryptophan, Antibiotics (Ampicillin, Spectinomycin,
Chloramphenicol)
18 60mm Petri Dishes
3 6-well Light chambers
3 Power supplies (12V)
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Appendix C: Bactograph Experiment Assessment
Part I: Please check the box that most closely matches your response for each of the following statements:
Strongly
Agree
1
The kit was easy to work with
2
The experiment produced a photograph resembling
the image on the applied mask
3
The experiment could be performed within one
hour
4
I enjoyed performing the experiment
5
I felt 100% engaged while performing the
experiment
6
I understood the concept of bactography
7
I could identify the materials I needed from the kit
8
As the instructor, the protocol was easily
understandable
9
My students could understand and perform the
protocol
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Agree
Not sure
6
Disagree
Strongly
Disagree
Part II:
Check YES or No for each of the following:
Yes
9
No
My Lab has the following equipment or materials:
Incubator
Hot Plate
Sterile Volumetric Pipettes
60-mm Culture Plates/Petri Dishes
10
I was able to find an alternative for each of the following:
Incubator
Hot Plate
Sterile Volumetric Pipettes
60-mm Volumetric Pipettes
Part III: Please answer the following questions:
1. Which part of the experiment was easiest/ most difficult?
2. What alternatives of the items listed in question 9 (above) could be/were used?
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3.
What modifications to the experiment would you suggest to better benefit participant learning and/or participation?
4. What parts of microbiology were/could be taught through the bactograph kit?
5. Which pre-culture method did you use? Which method produced the best result?
6. Do you think the bactograph experiment could be performed as part of your curriculum? If not, why?
7. Is there anything else you would like added to the bactograph kit or experiment?
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