Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 61
Step 7. Streaking E. coli bacteria
Now that you have made your LB agar plates, you can grow bacteria on them. The Canvas Kit is meant for you
to make bioart. By painting bacteria onto the LB agar using various utensils and then incubating them, you can
create a living painting. As you learn to become a Genetic Engineering Hero, you will also practice a method of
putting bacteria on an LB agar plate called streaking.
There are different types of streaking that you will learn as you follow the Zero to Genetic Engineering Hero Jour-
ney. Today’s streaking is simple. Your goal is only to transfer some of the colored bacteria to your LB agar petri
dish so that you have a greater quantity of cells (bacteria paint) that are freshly grown to use in your experiment.
On your one plate, you want to create a painting palette of bacteria paint that you will use as your “source” paint
for your bio-art (living painting). To do this, you will streak out cells using a procedure that you learned and
practiced in Virtual Bioengineer Canvas Kit Edition and that you can see step by step in the instruction manual:
1. See how many bacteria paint colors are included in your Canvas kit. This will typically be 3 colors. Using a
marker, divide the bottom of your petri dish in the corresponding number of sections and label the sections
with one section for each bacteria paint color you have.
2. Get a yellow inoculating loop. Dip it into one of the bacteria paint tubes. Inspect the loop to see if it looks
“wet,” indicating that you’ve dipped into the cells/agar. Then trace a zig-zag in the rst section of your petri
dish. Discard the loop in your inactivation bag.
3.
Using a new yellow loop each time, repeat the above step for each tube of bacteria paint. Always discard
your loops in your inactivation bag.
Step 8. Incubating E. coli cells
Turn on the Incubator of your DNA Playground Minilab to 37 ˚C. As the incubator heats up, you should ip your
LB agar plates so that the bottom is up and the lid is down. Following the manufacturer’s instructions, place the
ipped LB agar plates with E. coli bacteria onto the paddle, cover with the humidity chamber, then place into
the incubator. Turn on the timer to better monitor growth. Incubate the cells for 12 to 24 hours (but up to 48
hours if needed). You will start to see them after ~12 hours, and they will start to change color shortly after. You
are now growing your rst bacteria on your journey to becoming a Genetic Engineering Hero. Congratulations!
Figure 3-8. Step 6. Following the stencil example in the instruction manual, divide and streak one of your plates with the bacteria
paints (colored bacteria) included in the kit.
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 62
Step 9. Viewing plates of grown bacteria
Over the incubation, a single bacterium will grow and divide many times to form a colony. On your streaked plate,
you should see something similar to what you saw in Virtual Bioengineer Canvas Kit Edition. There will be areas
of high concentration of bacteria and areas where you can see single dots. These dots are called bacteria colonies!
In this exercise, all of the bacteria on your LB agar plate should be the same color or colors as written on the tubes
of cells you received in the kit. E. coli have a smooth, glossy morphology and an overall circular shape (Figure 3-9).
If you see growth on your LB agar plates that is:
• A different color than you expect, this is likely contamination from your environment.
• A fuzzy growth is likely mold that was in the air
• An irregular shaped colony (not a circle like E. coli colonies are) this could be skin bacteria that you trans-
ferred into your experiment.
If you see any contamination in your plates, you should put them in your inactivation bag immediately so they
can be inactivated at the end of your experiment (Step 13).
Once you see your results, you can move onto using these colored bacteria as your paint for the bioart you will
make on your remaining petri dishes.
When creating your paintings, you will also be practicing a new skill called re-streaking bacteria. Re-streaking is
used to grow more of an experiment’s results for further experimental needs. In your case, this means that you
will use the grown bacteria on your incubated plates as the ‘source’ bacteria for streaking and creating bioart on
your remaining plates, instead of using the cells in the tubes that came with your Canvas Kit. Note that typically
when you use re-streaking in a laboratory experiment, you only touch your inoculating loop to a single colony,
but for painting, you can touch many colonies at once and/or the lawn of bacteria.
Note that if nothing grew on your petri dish, if the colonies did not change colors as expected or if your exper-
iment was contaminated, discard the plates in the inactivation bag and repeat Step 7 & 8 on a new petri dish
using the tubes of bacteria paint (cells) as your source bacteria. Remember to make sure to dip the yellow loop
Figure 3-9. E. coli cells engineered to make cyan uorescent pigment, streaked onto a selective LB agar plate, photographed under black-
light (cyan bacteria will appear white under natural light). Characteristics of a successful experiment include: i) All bacteria on the plate
are the expected color; ii) E. coli form circular, smooth and glossy colonies; iii) A lawn of E. coli forms a solid, smooth, glossy mass of cells.
Lawn of E. coli
Colonies of E. coli
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 63
into the tube of bacteria paint in order to transfer the bacteria to your LB agar plate. You should be able to see
some trace of your loop’s trajectory on the surface of the agar if you hold it in the light. It will look like a “wet”
trace. The individual bacteria themselves are too small to see and will require time to grow into colonies for you
to verify success.
Growing E. coli Going Deeper 3-3
Humidity: Bacteria need water/humidity to operate. The cell machinery and chemical reactions that occur
in E. coli require the presence of water. All of the food, minerals, and salts in the cells are dissolved in water,
enabling them to move and interact with other cellular molecules such as lipids, sugars, proteins, and DNA.
If the cells dry up, then cells enter “suspended animation” or break apart and die. In times of stress, such as
dehydration, some bacterial species other than E. coli enter a long-term survival mode where they sporulate,
becoming a spore that can remain in suspended animation for thousands if not millions of years.
Flipping plates so the bottom is up is a conventional technique for preventing LB agar plates from drying
out. In the warm incubator, water in the LB agar evaporates. When a plate is inverted, that evaporating water
does not escape as easily. It also keeps the surface of the LB agar moist. This aids in bacterial growth.
Temperature: Thermal energy (temperature) has a signicant inuence on the activities happening within
cells. A cell’s temperature can inuence how uid the cell membranes and cellular components are, speed-
ing up or slowing down chemical reactions. A change in temperature can have profound effects on how
fast an organism grows, along with the processes that go on within a cell. As you will see in later chapters,
temperature can play a role in whether parts of the DNA blueprint (e.g. genes) are read by the cell. You can
try growing your cells at 30˚C instead of 37˚C to see the difference in how fast the cells grow!
Why do you grow E. coli at 37˚C? E. coli bacteria have evolved inside of the large intestines of mammals like
humans. Because our body temperature is 37˚C, the microorganisms have evolved to grow most efciently
at this temperature. In optimal conditions, E. coli will divide every 20-30 minutes. That’s about twice every
hour. After 12 hours (about 24 divisions) a single bacterium could divide into 16,777,216 bacteria. However,
because of space and food constraints, as bacteria grow more densely, the pace of division slows.
Step 10. Painting living art (bioart) with E. coli bacteria
Figure 3-10. Bioart can be made by painting colorful bacteria onto an LB agar petri dish. Use the bacteria paint you streaked and
incubated as your ‘paint’ for your art.
Canvas
Canvas
Canvas
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 64
After incubation, verify that the bacteria paint has changed color before starting this step. If it is not yet
colorful, incubate longer or refer to the manufacturer’s instruction manual. The colors develop best at
37˚C. If you are using a DIY incubator where the temperature is not 37˚C or very close to it, your colors may
not develop fully - they will stay in shades of pastel. You can use them as pastels paint.
Take two LB agar Petri dishes (from Step 1) out of the refrigerator. For each petri dish:
1.
Draw your own design on one of the blank stencils. Place one of your unused LB agar plate on top of your
stencil or the pre-made image stencil. Simple images without many details work best for the rst time!
2.
Take your petri dish streaked with colorful bacteria from your incubator. This is your painting palette. Using
your ‘bacteria paintbrushes’, dip one into the paint on your painting palette. Paint your art by carefully
streaking across the surface of the LB agar of your new plate, following your image stencil. When doing bioart,
it is okay to dip your painting instrument into the bacteria more than once, but since you will be making
more painting s on remaining petri dish(es) keep some paint and paintbrushes for later.
Remember to verify that your bacteria paintbrush touched the paint. After you have painted your image on the
agar, you should be able to see some evidence of your work as a ‘wet’ trace if you hold the petri dish in the light.
The individual bacteria themselves are too small to see and require time to grow for you to verify success. Paint-
ing with bacteria is hard because it is like painting with invisible ink that appears days later!
3. You can choose to paint another agar plate right away or save it for later. After you are done painting for the
day, put used items like loops and cotton swabs in the inactivation bag. Close your painting palette petri dish
and place it in a resealable bag (You can use the same one that has the unused agar petri dishes). Place it,
along with the remaining unused petri dish, and bacteria paintbrushes in your lab refrigerator or in a sealed
container that goes in the refrigerator (Chapter 2). You will incubate your painted plate(s) in the next step.
Step 11. Incubating E. coli cells
Once again, you will be incubating petri dishes. This time, you will incubate your painted petri dish. Turn on
the Incubator of your Minilab to 37˚C. As the incubator heats up, you should ip your LB agar plates so that the
bottom is up and the lid is down. Following the manufacturer’s instructions, place the ipped LB agar plates
with E. coli bacteria onto the paddle, cover with the humidity chamber, then place into the incubator. Turn on
the timer to better monitor growth.
Incubate the petri dishes for ~24-48 hours. You will start to see growth after ~12 hours, and the bacteria will
start to change color shortly after.
Step 12. Viewing & Preserving your bioart with a Keep-it Kit
Figure 3-11. Left: Bioart made with uorescent bacteria by Nathan Shaner, photo by Paul Steinbach. Created in Nobel laureate Roger
Tsien’s lab. Right: Bunny bioart made with Amino Labs’ Canvas kit under black light and natural light.
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 65
After 24 to 48 hours of incubation, have a look at your work. You’ve created bioart! Since you have one LB agar
plate remaining, you can repeat the painting exercise on this plate using what you learned after seeing your
bioart results. Bioart is not an easy art form to master!
If you have a Keep-it Kit, you can now download the instructions and complete the Keep-it Kit exercise. As part
of this exercise, you will let your plates dry so that there is very little moisture on the surface of the petri dish.
This will be followed by embedding your cells in a clear resin and allowing it to harden over 24-48 hours.
Step 13. Clean-up and inactivation
As you were going through the exercise, you placed used materials into the inactivation bag. When cleaning up
at the end of this experiment, you should place all of your materials that have come into contact with bacteria
into the inactivation bag, including your plates (don’t forget to photograph them before destroying/inactivating
them!). Note that the paper packaging for the inoculation loops can be disposed of in the regular garbage. You
can also put your used gloves into the inactivation bag if you believe they’ve come into contact with the bacteria.
All of your tubes should be placed in the inactivation bag with the lids off so the bleach water can enter them.
Follow the steps provided by the manufacturer:
1.
If you haven’t set up your discard container (Chapter 2), place your inactivation bag into a bowl, bucket, or
similar type container to hold it upright and to contain any liquid if you accidentally puncture the bag.
2. Use your ‘sterile water’ bottle after Step 6. Add one full bottle (250 mL) of concentrated bleach to the bag. Be
careful not to spill any bleach on yourself as it is caustic. Wear safety glasses & gloves!
3. Add 4 to 6 “sterile water” bottles of warm water (total of ~1 L) to the bag. The goal here is to submerge all the
materials so that they are all in contact with the bleach water solution.
4. Seal the bag tightly and gently massage or swirl the items to ensure they are in contact with the bleach.
5. Let it stand for 24 hours or longer. This will inactivate the samples. In other words, it will kill the bacteria and
break down DNA.
6.
Afterward, empty the liquid in a toilet by clipping a corner of the inactivation bag with scissors (while over
the toilet) and place the solid waste in the trash can. Cut only a small hole in the corner of the bag so that the
solid materials do not escape.
Figure 3-12. Use the inactivation bag to safely dispose of your experiment materials.
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