152 Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
S
H
3
C
O
+ +
H
3
C
O
R
OH
O
R
CoA CoA
acetyl-CoA alcohol CoA ester
Figure 6-5. Atf1 enzyme takes an acetyl group from acetyl-CoA and transfers it to the isoamyl alcohol.
B.
Place your IA substrate tubes in your centrifuge and “pulse” spin them down for 10 seconds. This will
ensure that all the substrate is at the bottom - This is how Genetic Engineering Heroes guarantee all of their
samples are at the bottom of the tube.
C. Create your selective LB agar as you have previously, but DO NOT POUR IT YET! Bring your sterile distilled
water to a rolling boil. Add your LB agar powder and swirl to dissolve. Heat it further in 4-second intervals
until you see the molten LB agar foam (boil). Add your antibiotic and swirl to dissolve it as you have done in
prior exercises. DO NOT POUR!
D. While your LB agar is cooling, you must complete this step rather quickly. Using the pipet included in
your kit, push the paper disks to the side of the three petri dishes. Using the pipet, transfer an entire tube of
IA substrate to the middle of one of the plates and then using the pipet push the paper disk over the top to trap
the IA underneath. Complete the same action with the other disks, tubes of IA substrate, and plates.
E.
Pour your selective LB agar into each plate. Use a sterile yellow loop to hold the paper disk on the bottom of
the IA dishes when you pour in the LB agar - the disks may begin to oat, but you can keep them on the bottom
of the plate by holding them with a yellow loop. You may use the same yellow loop for all plates. Allow your
dishes to cool and solidify. The paper disk will help to release the IA substrate into the LB agar slowly. This slow
release will create a sustained overripe banana smell that will last between 1-3 days.
Step 5. Culture engineered cells on your selective LB agar plate
If your DNA Playground incubator has 8 plate capacity, you can streak and incubate all four plates at the same
time. If you have a two plate incubator, you can streak/incubate two of four plates in any order - the cells will
grow within 24 hours, after which you can streak and incubate the next two plates.
Following the plate labels you previously made, use the dual streak method (Chapter 5) for culturing cells to
ensure you have a lot of cells growing across the surface of the plate. As the IA substrate diffuses to the surface,
the cells will absorb the substrate, and the expressed enzymes will process isoamyl alcohol into a different
molecule called isoamyl acetate - which has the smell of overripe (sweet) bananas.
As you open the lids of the plates, the smell will disappear and you will need to wait for it to replenish. Try to
do a blind smell test by enlisting someone’s help!
ATF1 Banana Smell Going Deeper 6-1
The protein enzyme that you have engineered the K12 E. coli to produce is called alcohol acetyltransferase
1, abbreviated Atf1. Atf1 catalyzes a chemical reaction whereby the musky smelling substrate, isoamyl
(smell)
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153Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
No color No color Color
HO OH
OHOH
OH
O
ß-Galactosidase
X-Gal
+ H
2
O
HO
Cl
Br
N
H
O
Cl
Br
N
H
O
Cl
Br
N
H
Figure 6-6. Beta-galactosidase removes a sugar ring from X-gal with the help of a water molecule. The products then “dimerize
into a colorful molecule.
alcohol, is converted to overripe banana smelling isoamyl acetate (Figure 6-5). When you added the isoamyl
alcohol substrate underneath the LB agar, it slowly diffused into the LB agar, all the way to the surface where
your engineered cells are. The substrate then crossed the outer membrane, intermembrane space, and
inner membrane of the cells into the cytoplasm where many Atf1 enzymes were expressed. The chemical
reaction includes three essential players:
• isoamyl alcohol substrate
• acetyl-CoA substrate (already created by the cells and is residing in the cytoplasm)
• Atf1 enzyme that catalyzes the reaction
Atf1, isoamyl alcohol, and acetyl-CoA complete the Four B’s around the cell. When both substrates bind to
the Atf1 enzyme, a chemical reaction occurs. A video describing how both substrates are changed by the
chemical reaction can be found below. In short, a small chemical acetate group is transferred from the
acetyl-CoA to the isoamyl alcohol. The products of the reaction include a CoA and the isoamyl acetate ester
(Figure 6-5). You can nd an explanation of the Atf1 chemical reaction at https://amino.bio/atf1
By adding the small acetate group on the isoamyl alcohol, you substantially change the characteristics of
the molecule - it now makes a noticeably different smell!
Usually, this reaction doesn’t happen without ATF1, so did you notice a difference between your ATF1+IA
plates compared to your negative controls? Also, have a look back to Going Deeper 3-7 to refresh your
memory about how enzymes help to make chemical reactions happen.
Exercise 2: Enzymatic processing to generate color
In Exercise 1, you engineered K12 E. coli cells to create Atf1, a protein enzyme that is capable of modifying alcohols
into esters. This changes the chemical structure and also the physical odor properties of the molecule.
In Exercise 2, you will complete another enzymatic processing experiment, except this time using the cell extract
rather than in a petri dish. In this experiment, you will be engineering E. coli cells to express an enzyme called
beta-galactosidase. This enzyme is useful for processing a certain substrate molecule that does not have any
color into colorful molecules. The substrate in this reaction is typically called X-gal, where the “X” can be one of
many colors, and the “gal” standing for galacto. Two X-gal molecules are included in the Blue-It Kit: Blue-gal with
its chemical name 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside, and Yellow-gal with its chemical name
4-nitrophenyl β-D-galactopyranoside. Visit amino.bio to see if new colors are available!
In the reaction, the beta-galactosidase enzyme binds a water molecule to X-gal and removes a sugar from the X-gal
molecule. When enzymes use water to complete a chemical reaction, it is called hydrolysis. Two of the hydrolyzed
X-gal molecules then dimerize (come together in a pair) resulting in the molecules’ physical properties changing
and
becoming colorful (Figure 6-6).
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154 Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Step 1. Download the instruction manual for the Blue-it Kit
Find the manual at https://amino.bio/instructions.
Step 2. Complete genetic engineering and extraction procedures
Similar to Chapters 4 and 5, complete the genetic engineering using the Blue-it Kit to obtain colonies of E. coli
genetically engineered to express the beta-galactosidase enzyme. Using the cultured genetically engineered
bacteria, complete the extraction to obtain a tube of sterilized cell extract containing the beta-galactosidase
enzyme that you engineered the cells to express. A new and important aspect of this extraction is that the
beta-galactosidase is sensitive to oxygen when outside of the cells and so you need to add a special additive to
your Lysis Buffer. Called a “reducer, dithiothreitol (DTT) is a small molecule that has a sulfur group that makes
DTT able to combat the harm that oxygen can have on beta-galactosidase and keeps beta-galactosidase from
breaking apart. In your kit, you’ll create a tube of DTT, called Enzyme Stabilizer, by combining the DTT powder
in the Enzyme Stabilizer tube to the Enzyme Stabilizer Dissolving Buffer. You will use the Enzyme Stabilizer
twice, once in this step and once when catalyzing your reaction. Make sure you put your tube of Enzyme Stabi-
lizer in the freezer after this step.
You’ll denitely notice that DTT is smelly and this is due to the sulfur atom that is part of the molecule! As
Genetic Engineering Heros do, spin your DTT powder tube in the Enzyme Stabilizer tube in a microcentrifuge
to ensure that all of the DTT powder is at the bottom of the tube. Next, add the Enzyme Stabilizer Dissolving
Buffer using a small pipet. Pipet up and down a few times to mix, until the powder is dissolved.
As per the instruction manual, add your Enzyme Stabilizer to the Lysis Buffer. Then, scrape and add your cells
to the Lysis Buffer tube. Add the Lysis Accelerator as you have in the past. Incubate as recommended in the
instruction manual.
After incubating your lysis reaction, microcentrifuge your sample and complete the lter-sterilization as you
did in Chapter 5. You should now have a tube of sterile beta-galactosidase enzyme extract, also called your cell
extract, for use in your experiment. Note that you will also need some of this cell extract for the post-practice
exercise where you make pure oxygen from hydrogen peroxide. You will not be using all of it for the Blue-it Kit
reaction so store the tube of cell extract in the refrigerator after using it in the next step. Further note that you’ll
want to complete the post-practice exercise within one day of refrigerating your cell extract.
Step 3. Dissolve your substrates
Within the Blue-it Kit, there are two tubes labeled “Blue-gal Substrate” and “Yellow-gal Substrate”. These
tubes have a small amount of white powder in them, and you rst need to dissolve the powders with Reaction
Buffer. Reaction Buffer is mostly water but has the right pH (acidity) to make sure the enzymatic processing
works well, and the enzyme stays stable.
Similar to Step 2, you’ll need to add Enzyme Stabilizer to each tube of Reaction Buffer.
The Blue-gal and Yellow-gal are shipped to you in powder form because they are not very stable when dissolved
in a liquid. This is why you will dissolve them in the Reaction Buffer just before adding in your cell lysate. Simi-
larly, the DTT is not very stable in other liquids, and that is why you dissolve it just before you complete the
experiment. Refer to the instruction manual to complete this step.
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155Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Step 4. Add cell extract beta-galactosidase to the substrate
Get ready for chemical wizardry! Using a pipet, add a small amount (50-100 uL) of cell extract to each substrate
tube. The plastic pipet will have a small 100 uL marker on it to help guide you. Place the lids on rmly and
invert them to mix them thoroughly. Place the tubes on your DNA Playground Hot Station set to 37 ˚C and sit
back and let the beta-galactosidase do its job! Over the course of the next 24 hours, the samples will become
increasingly blue and yellow. You’ll see yellow start to appear in minutes (usually within 10 minutes), and the
blue will appear over the course of hours.
Enzymatic Processing Going Deeper 6-2
You have now completed enzymatic processing! In both exercises, you engineered the cells to produce
protein enzymes (Atf1 & beta-galactosidase) that were able to complete the Four B’s and ultimately interact
with substrate molecules (isoamyl alcohol + acetyl-CoA or Xgal+H
2
O) and turn them into products. But why
did you do the Smell-it Kit within the cells vs. the Blue-it Kit as an extract? These are both useful methods
and choosing between them depends on the many factors such as the stability of the protein enzymes, the
availability of the substrates in the cell, and the cost of buying substrates.
In Exercise 1, a key reason for using streaked cells on plates, rather than an extract, was that one of the
two substrates, acetyl-CoA, is very expensive to purchase and you needed the cell to keep microfacturing
it naturally for your chemical reaction. Because of this, lysing the cells would have stopped the cells from
making more acetyl-CoA, and you would have had only a small amount of acetyl-CoA for the reaction (when
you lyse the cells, they will not create any more acetyl-CoA). Doing the reaction in living cells means the
cells continue to grow and they can keep producing acetyl-CoA, and so long as you have lots of IA available,
the reaction can continue to occur. This means you could keep adding IA to the cells and they would keep
processing it into isoamyl acetate! For reference, 0.1 grams of acetyl-CoA costs more than $1000 to buy!
Including it in the kit would sure increase the cost of the Smell-It Kit! Let the cells make it for you!
In Exercise 2, only one substrate molecule is needed for the chemical reaction, and that chemical is reason-
ably priced. There is no need to have the cells continue growing to create a substrate. Because a single
beta-galactosidase enzyme can cause the chemical reaction to happen thousands to millions of times
during its lifetime, you can simply use the extract with the engineered enzyme and add the X-gal substrate.
An alternative protocol for Exercise 2 could involve growing cells in a petri dish, similar to Exercise 1. Pour-
ing or pipetting a small amount of the X-gal substrate over the top the plate of cells, or adding it into the
LB agar when you pour the plates is common. The substrates in the LB agar can cross into the cells. The
enzymes inside the cells will begin catalyzing the same chemical reaction inside of the cells - the colonies
of bacteria would turn from white to colored.
Naming Enzymes Pro-tip
Protein enzymes are usually easy to spot because their name will end in “…ase”. Generally, the beginning
prex of the enzyme name will give you some historical or chemical context about what the enzyme does,
and then “ase” sufx is added to the end.
In Exercise 1, alcohol acetyltransferase (Atf1) is a great example. One might deduce from its name that
it binds alcohols and transfers an acetyl group to it. In Exercise 2, the enzyme is beta-galactosidase. One
might deduce that it can catalyze reactions involving beta-galactoside sugars. And if we think even further
back to Chapter 4, RNA polymerase was the enzyme that connects ribonucleotides into a polymer.
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156 Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Create Pure Oxygen! Breakout Session 1
Using your Blue-it Kit cell extract (after you do the
lysis, centrifugation, sterilization you will have your
Blue-it Kit cell extract) you will attempt to create
pure oxygen. If you extract is old (more than 1 day)
and has not been stored in the refrigerator, the
enzymes may have lost function. In this case you will
have to try again with fresh cell extract.
Have you ever had a scrape or cut in your skin that
you disinfected using hydrogen peroxide? Hydrogen
peroxide is very harmful to bacteria. This is why it
is used as a disinfectant. K12 E. coli naturally create
a protein enzyme called catalase (Figure 6-7). This
enzyme is part of the bacteria’s natural defense
system. If the bacteria encounters Reactive Oxygen
Species (ROS), catalase can bind to and convert those
molecules into less harmful molecules.
In this post-practice exercise, we’re going take advantage of the fact that the enzyme catalase is naturally
produced in E. coli, and that there will be thousands of catalase proteins within your Blue-it Kit cellular
extract from this chapter. We’re going to use catalase to convert hydrogen peroxide (harmful to E. coli)
into pure oxygen (not harmful to E. coli).
The chemical reaction that catalase catalyzes is the following:
2 H
2
O
2
(l)
—catalase—> O
2
(g)
+ 2 H
2
O
(l)
This means that catalase will convert two molecules of hydrogen peroxide (H
2
O
2
), into one molecule of
oxygen gas (O
2
) and two molecules of water (H
2
O).
1. In a transparent container such as a beaker, drinking glass or jar, create a 1/3 solution of hydrogen
peroxide with distilled water (tap water is OK, it just may not work as well). You can do this by adding
1 part hydrogen peroxide (e.g., 1/3 cup) to 2 parts water (e.g., 2/3 cup). You can use these measure-
ments, or make as much or little of this mixture as you like.
2. With one of the pipet from the Blue-it Kit, transfer a small amount (~100 uL) of your cell extract into
the hydrogen peroxide mixture. The small line indented on the pipet marks 100 uL.
3. After you’ve added some cell extract, wait a few minutes, up to a few hours, and you will start seeing
magic happen. Bubbles will begin to form and rise to the surface. Bubbles will also begin to form
on the edge of your container. These bubbles are pure oxygen gas that comes from the chemical
reaction that the catalase enzyme is catalyzing!
[Raw material [Substrate(s)] —> Finished Product [Product(s)]
This is a very common theme among manufacturers such as large steel mills that take in raw materials
such as iron ore and rene it through various processing into nished products such as steel beams
that are used for building. Pharmaceutical companies use enzymatic processing to process less useful
molecules into real medicine.
Figure 6-7. Ribbon diagram of Catalase. Catalase is a pro-
tein enzyme that can convert reactive oxygen species into
less harmful molecules. It is naturally produced in E. coli
bacteria. Source: Protein Data Bank (PDB): 1iph, illustra-
tion created with Chimera.
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