96 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
At the end of this step, your transformation buffer should be cloudy because of the opaque cells suspended in
the clear liquid. If you see clumps of cells oating around you should mix them further by blending vigorously.
Imagine it’s a cold winter, and you’re going to freeze - start the re!
Once you have a cold Transformation Buffer that is cloudy with cells, your cells have become chemically compe-
tent. Your cells are immediately ready for the transformation. Do not wait more than 3 minutes after adding
your cells to the T. Buffer before moving onto the next steps.
Step 7. Add DNA plasmids and Heat Shock
Your cold competent cells are ready for you to add DNA plasmids into the mix.
A. Tap the DNA tube on the table so the liquid collects at the bottom of the tube. Dip a new 1 uL blue loop in
the tube of DNA. Swirl and spin the loop for about 10 seconds to ensure the DNA is mixed and that the center
of the loop becomes lled with liquid.
Pull the inoculating loop out of the DNA tube and check to see if there is liquid in the center of the loop. Remem-
ber blowing soap bubbles? You should see something similar, like the lm of soap in a bubble blowing loop. If
you can see liquid in the center of the loop, you’re ready to add it to your chemically competent cells!
B. Add DNA to your cells in the T. Buffer tube and incubate: Keeping your cells chilled on the Cold Station, dip
your inoculating loop with DNA into your chemically competent cells. Swirl and mix in the DNA for 10 seconds.
Remove the loop and discard it. Let your cells and DNA remain on cold for 5 minutes. During this time the DNA
will begin binding to and interacting with the cells. If you haven’t yet, turn the hot station to 42˚C, you will need
it in the following step.
C. Heatshock the cells: It is now time to get your DNA into the cells. In this step, you will change the tempera-
ture of the cells from ice cold (4˚C) to hot (42 ˚C), then back to cold. This is called a heat shock because you are
shocking the cells with warm temperature. During the shock, the DNA enters the cells.
Transfer the tube of cells and DNA to the 42˚C Hot station. Turn the timer on, wait for 90 seconds. After 90
seconds, return the tube to the Cold Station (4˚C). Let it stand for 2 minutes. This traps the DNA inside the cells.
During this short incubation, you can now set the Hot Station to 37˚C; you will need this temperature in the
next step. The Hot Station will slowly cool down and if it does not reach 37˚C by the time you are ready for the
next step, that is ok. You can continue onto the steps as it cools down.
Figure 4-10. Step 7 A. Dip a new blue
loop into the DNA tube - make sure you
can see liquid in the loop after you dip!
Figure 4-11. Step 7 B. Mix your DNA plasmid
in the T. Buffer and cell solution.
9m
20s
Figure 4-12. Step 7 C. Heatshock your
cells, T. Buffer and DNA.
90s
2m
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97Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Plasmid Going Deeper 4-4
What is a plasmid? In the What is DNA? you learned that a DNA plasmid is a circular double-stranded helix
of DNA. You can imagine DNA plasmids as small DNA molecules that are separate from the cell chromo-
somal DNA, and that can replicate independently. The DNA plasmid has important sequences and genes
that the cell reads once the plasmid enters the cell (Figure 4-13). These are:
Selection marker gene: The Selection marker gene is often used to help select for bacteria that you’ve
engineered while growing the bacteria in LB agar. During the engineering process, a very small number of
your competent cells actually take up the DNA plasmid. By giving the engineered bacteria a ‘superpower’
like antibiotic resistance with the DNA plasmid, you ensure that only your engineered bacteria - cells that
have taken in, read and are executing the DNA plasmids successfully – can grow on selective plates with
specic antibiotics. Bacteria that didn’t take in DNA plasmids (and are therefore not engineered) don’t have
antibiotic resistance. These cannot survive the antibiotics and die. In simple terms, engineered bacteria
grow, and non-engineered bacteria do not.
Origin of replication (ori): The ori is a sequence that is recognized by the cell machinery and tells the cell
to copy the DNA Plasmid. This is really important because, without the ori, the plasmid would not get copied
and divided as the cells divide! An ori is like the rst lines of computer code that specify what libraries and
sub-programs (the DNA plasmid) to “include” in the program.
Trait gene: Many different kinds of products can be ‘microfactured’ by engineered cells. In addition to
adding the selection marker gene, another gene is typically added to cause the expression of a new and
interesting trait. Usually, this is the trait that tells the cell machinery to produce what the genetic engineer
is looking to make. For example, the colored pigment in your Engineer-it Kit.
Negative charges: In Chapter 1, you learned that DNA is negatively charged due to the phosphate (PO4
-
)
molecules that make up its backbone. In Chapter 3, you learned about the lipopolysaccharide (LPS) slime
layer and phospholipids (Figure 3-21) that make up the outer surface of E. coli bacteria. This outer layer is
negatively charged. This is primarily because of the charged head groups of the lipid bilayer. Look back to
Figure 3-22 in Chapter 3 and look at the negatively charged group of the example lipid - it is also a phos-
phate! What happens when two negative charges come into contact? They repel. Now, imagine what might
happen if a positively charged ion, like calcium (Ca
2+
) was present... Your Transformation Buffer is mostly
made up of sterile water and Ca
2+
. One hypothesis as to why T. Buffer helps to get DNA into cells is that the
Ca
2+
ion is able to bind both the negatively charged surface of the cell as well as the negatively charged
DNA. In a way, Ca
2+
acts as a ‘glue’ that binds to both, causing the DNA to get close to the cell. This is called
a ‘coordination complex’ (Figure 4-14). When the DNA gets cozy with the cells, it increases the chances of
the DNA getting into the cells during the heat shock.
Cell
Ca
2+
Figure 4-14. Coordination complex. Ca
2+
ions bind to DNA
and the cell’s outer membrane, acting like glue between the
negatively charged molecules.
Figure 4-13. A DNA plasmid
~3,000 nt/bp
S
E
L
E
C
T
I
O
N
/
G
E
N
E
T
R
A
I
T
/
G
E
N
E
O
R
I
~3,000 nt/bp
S
E
L
E
C
T
I
O
N
/
G
E
N
E
T
R
A
I
T
/
G
E
N
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O
R
I
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98 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Step 8. Recovery step
The heat shock step was harmful to the cells, so immediately following the cool down period of the heat shock
step, you must ‘recover’ them.
You will do this by adding Recovery media to the cells. Take the lid off both tubes and pour the Recovery
Media into the tube of cells and DNA, then close the lid. Note that some liquid will likely stay in the bottom of
the Recovery media tube. This is OK, just transfer the majority of the liquid!
Mix the cells in the Recovery media by shaking them. Then, bring all the liquid down to the bottom by either
doing the ‘Whip-it’ maneuver (https://amino.bio/whip-it) or by tapping it on the table.
Place the tube of transformed cells into the Hot Station and set to 37˚C. For the best results, let your cells
recover for 12 to 24 hours. This not only allows them to start growing and dividing again, it also gives the cells
the opportunity to start expressing the antibiotic resistance gene. This is very important for the next step when
you spread your cells on the selective (E) plate that contains antibiotics.
Heatshock & Recovery Going Deeper 4-5
Heat shock: When cells are cold, the lipids in the membrane become more rigid and tightly packed. When
they are warmed, the membrane becomes more uid and permeable. Imagine making bacon on the stove.
As you’re cooking, a liquid grease comes out of the meat into the pan. After you have completed cooking and
the pan cools down, the grease cools and solidies. This phenomenon may be similar to what happens in the
membrane of the cells: the more uid the membrane, a better chance that DNA can cross the membrane.
As you’ll see in Chapter 6, this is due to special bonding in the hydrophobic tails of the lipids.
Another analogy is a spa treatment. Often when getting a skin treatment, warm water or steam is used to
open up the pores of the skin so that they can be cleaned. After the cleaning, the skin is cooled in order
to close the pores. Perhaps a transformation is nothing more than a micro spa treatment - when you heat
shock the cells, pores in the membrane form so that DNA can enter into the cells. Upon cooling, the pores
contract or disappear and the DNA is trapped inside.
Recovery: Recovery media is LB liquid growth media. The LB agar powder you used to make your petri
dishes is similar to the recovery media except LB liquid growth media simply doesn’t have any agar. This
allows you to grow cells in a liquid broth environment, rather than on a solid gel substance. LB has all of
the nutrients and minerals that E. coli bacteria need to start growing and dividing again. Just like you prob-
ably enjoy a nice meal after some hard work to help you recover your energy, the E. coli cells are enjoying
LB media. LB is their food source, after the hard work of becoming chemically competent and welcoming
DNA into their membrane.
Figure 4-15. Pour the Recovery media into the T. Buffer. Mix thoroughly and keep at 37 °C for a minimum of 1 hour, up to 24 hours.
45m
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99Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Figure 4-16. Step 9 A. (left) Plate your recovered cells, DNA and T. Buffer solution. Once it dries, ip before incubation. Step 9 B.
(right) Use a yellow loop to streak the Positive cells on your S(+) selective plate.
+
Note! In preparation for Chapter 5
Once you see your S(e) plate result, place it in a ziplock style bag in the refrigerator. You will want it in the next
chapter’s excercise! If you preserved your S(e) plate with a Keep-it Kit, that’s ok. A tube of pre-engineered cells will
be waiting for you in the kit for Chapter 5. But if you want to use your own engineered cells, don’t wait more than one
week before starting the Chapter 5 exercise.
Step 9. Plating and incubating your cells
After your cells have recovered, you can ‘plate them’. This simply means that you will pour the cells and recov-
ery media mixture onto one of your selective LB agar plates and spread the uid across the plate. In this step,
you will plate both your experimental cells (e) as well as a positive control (+).
A. Pour ~1/2 of the recovered cells on your experimental selective LB agar plate labeled S(e). Using a new
yellow inoculating loop, gently spread the cells over the entire plate and let stand with the lid half-on until the
liquid evaporates. This can take up to 30 minutes, depending on your enviroment’s temperature and humidity .
If you’d like to speed up the drying process, place the plate on top of the Cold Station of your Minilab with the
plate lid half-on. Then turn on the Cold Station. The fan air will aid in evaporating the liquid. After the liquid has
fully evaporated, put the lid on, and ip the plate in preparation for incubation with your positive control plate.
B. While your S(e) experiment plate dries, nd the tube of positive control cells (+) from your Engineer-it Kit.
Dip a new yellow inoculating loop into this tube of (+) cells and zigzag the loop across the S(+) control plate,
just like you streaked the negative control. Place the plate lid back on, and ip it in preparation for incubation.
Place your S(e) and S(+) plates into the incubator at 37˚C for 24-48 hours. Over this time period, you will start
to see bacterial colonies grow. After about 16 hours you will see very small colonies that may or may not be
expressing your trait yet (such as a color pigment). At about 24 hours you will see the trait being expressed,
while at 48 hours and beyond the trait will become more pronounced. Your (+) control should grow and change
color quicker than your newly engineered cells.
Positive Control Going Deeper 4-6
A positive control is an experimental sample in which you expect a known response if the experiment is
running nominally. In this experiment, the positive control involves putting previously engineered cells
that have antibiotic resistance (+ cells), onto an LB agar plate that contains antibiotics. In this sample, we
expect that the engineered cells should grow, as long as the LB agar plates were made properly and have
the right amount of antibiotics and nutrients. If the cells do not grow, then this tells us that something may
have gone wrong when making the plates!
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100 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
on completing your third experiment - Genetic Engineering!
Step 10. What to expect & inactivation
After 24-48 hours of incubation, if your experiment was successful, you will see colonies (dots) of engineered
bacteria. Remember that getting a single colony is a success! Many scientists doing academic or industrial
research often hope for a single colony.
If you get more than one engineered colony on your S(e) plate, this means you followed the procedure very well.
As you repeat this experiment, you will very likely get more colonies than you did this time, because you will
have practiced the procedure and like most things in life, practice makes perfect!
Figure 4-17. Did you get one colony or more on your S(e) plate? Congratulations! One colony or more is a success! The rst and last
photos are experiments results by Zero to Genetic Engineering Hero Junior Editors Pau (rst) and Patricia (last)
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