124 Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Growth Phases Going Deper 5-1
As you learned in Chapter 4, for transcription to start, sigma factors must bind to the promoter of a gene.
This then enables the RNA polymerase to bind and begin transcribing the RNA from the DNA template
strand. The sigma factor for the promoters controlling the transcription of Engineer-it Kit proteins is
expressed in cells during a particular time of their growth. This stage of growth is called the “Stationary
Phase” of cell growth, as seen in Figure 5-5.
The S-curve is a graph that is used to describe the number of E. coli cells (y-axis) as a function of time
(x-axis). There are three major phases of E. coli growth:
1) Lag phase: This is the rst phase where there are low numbers of cells, and the cells have abundant
nutrients to support their growth. The cells begin growing rapidly.
2) Exponential phase: This is the phase when cell numbers increase the fastest. They have abundant food,
space, and the temperature is optimal for metabolic processes. They have not begun sending each other
chemical signals to slow down.
3) Stationary phase: This is the phase when nutrients become limited, and the cells begin sending chemical
signals to other cells to slow their growth. During the stationary phase, cell division slows, and cells start
preparing for the possibility of starvation.
It is in this third phase that your cells will really begin to start expressing the trait that you’ve engineered
the cells to create - about 12-48 hours after you start incubating. The genetic engineer who designed
these genes did this intentionally to make sure the cells would survive the transformation, start growing
and then start to express the trait once the cells are abundant. If the genetic engineer had made the cells
immediately express the trait, they might not have had enough energy and nutrients to survive. Asking the
cells to create two other traits (antibiotic resistance and color proteins in this case) is quite taxing on their
metabolism. Once the cells start slowing down in the stationary phase, they expend less energy on growing
and can spare that to create the proteins and genes they have been engineered to express.
The cell metabolism has a delicate balance that genetic engineers must respect and experiment with when
designing plasmids.
Figure 5-5. The S-curve describes the growth phases that organisms go through.
Time
Lag phase Exponential phase Stationary phase
# of Cells
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125Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Step 7. Extraction: Collect cells and start the lysis
After incubating your cells for 24-48 hours, a lot of cell colonies or lawns should have grown across the two
petri dishes. The cells should now be expressing the desired trait, such as a color pigment.
Once they are producing their trait in a sufcient quantity (e.g. the color is very bright and saturated), collect
the cells using a yellow inoculating loop and suspend them in the tube of Lysis Buffer. Use the cell-and-buffer
mixing method you learned in the previous chapter. Continue collecting cells on the loop and mixing them into
the Lysis Buffer until most of the cells on your plates are suspended in the Lysis Buffer tube:
A. Open your tube of Lysis Buffer and Lysis Accelerator. Place them in a tube rack or use the stations on your
DNA Playground to hold them upright. Be careful not to spill!
B. Gently drag your yellow inoculating loop across the surface of the LB agar to collect the cells inside the
loop. Once the loop has lots of cells, dip it into the Lysis Buffer tube and twist the inoculating loop like a blender
to dislodge and mix-in the cells - just like you did when creating competent cells during the transformation
process. Be careful not to be too vigorous; you don’t want to splash liquid and cells around.
C. Repeat the scraping and blending process until you’ve collected the majority of cells from your two plates.
Blend the cells and buffer for a further 60 seconds to make sure they are fully suspended. This will help the
surfactant in the Lysis Buffer begin lysing the cells.
Lysis Buffer Going Deeper 5-2
The Lysis Buffer contains a ‘gentle surfactant’ called Triton X-100. As in Chapter 1, when you lysed fruit
cells with the surfactant SLS (from household soaps), here you will use Triton X-100. Triton X-100 is often
used because while it breaks down cell membranes, it will not break down and destroy the proteins you’ve
engineered the cells to produce. There are many different surfactants used for lysing cells, and Triton X-100
is one of the most widely used for extracting and isolating proteins.
Figure 5-6. 7B. Scrape and mix your engineered cells in the Lysis Buffer.
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126 Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Step 8. Extraction: Lyse the cells
Now that you’ve mixed your cells into the Lysis Buffer, they will begin to lyse. You will now introduce a new
component to help aid in the lysis of the E. coli cells, the Lysis Accelerator.
The active ingredient in the Lysis Accelerator is a protein enzyme called Hen-Egg-White-Lysozyme (HEWL).
HEWL is extracted and puried from the egg-whites of hens and has a unique function of being able to attack
and break open bacterial membranes.
A. Using one of the pipets in the Plate Extract-it Kit, transfer all of the cells slurry from your Lysis Buffer tube
into the brown Lysis Accelerator tube. You will be using this pipet again in a few minutes to transfer the slurry
back into its original tube. Rest the pipet inside the Lysis Buffer tube for now.
B. Once you have moved all the buffer over to the Lysis Accelerator tube, rmly close the lid of the Lysis Accel-
erator tube. Then vigorously shake it for 30 seconds to ensure that it is thoroughly mixed. Tap the tube on the
table to make all the liquid collect at the bottom of the tube.
C. Using the same pipet as in Step A, transfer your lysed cells into the Lysis Buffer tube. This will help you to
complete the centrifugation Step 9, as it is easiest to do using a clear tube.
D.
Let it stand at room temperature for 1 to 24 hours. You can also incubate them in the refrigerator if you
want to incubate for 24 to 72 hours. You can shake the tube throughout this incubation to improve cell lysis.
HEWL Going Deeper 5-3
During this incubation, both the surfactant and HEWL are at work. Similar to the principles that you learned
in Chapter 1, the surfactant is interacting with the outer and inner membranes of the cells, then binding
up the lipids and some proteins into micelles. Since Triton X-100 is not as aggressive a surfactant as SLS,
it is common to also use the enzyme HEWL to aid the process of cell lysis further.
Hen Egg White Lysozyme (HEWL), is a protein enzyme that is capable of binding to and breaking apart the
peptidoglycan mesh in the intermembrane space of K12 E. coli cells. Recall from Chapter 3 that peptidogly-
can is the amino acid-sugar hybrid that acts as a security layer in the intermembrane space of K12 E. coli.
This is what the HEWL attacks and degrades. By using both HEWL and Triton X-100, you’re able to gently
break down the membranes of the cells so that the proteins you have microfactured can spill out.
Interestingly, humans produce enzymes that are similar to HEWL. These enzymes are produced in our tear
ducts and skin and act as the rst line of defense against bacteria in our eyes and on our bodies.
24h
+
Figure 5-7. Step 8. Move your cells that are suspended in Lysis Buffer to the Lysis Accelerator tube, and back. Incubate.
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127Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Step 9. Extraction: Pellet the cell debris
Centrifuging enables you to separate materials of different densities. After the lysing process is complete, the
cells, along with cell debris in micelles and other aggregates, will be denser (have more mass per unit volume)
than smaller molecules that remain dissolved (like proteins and water molecules).
Microcentrifuges typically spin at 13,000 revolutions per minute (RPM) or more, which corresponds to an accel-
eration of more than 10,000 x g (italicized g is the force of gravity, not grams). This powerful force will cause
the denser molecules to fall to the bottom of the tube in a compact mass called a “pellet”. In this exercise, the
pellet will include micelles, macromolecules like lipids, bits of cell membrane, and even genomic DNA. Small
cellular components, such as proteins and DNA plasmids, may not be pelleted and can remain in the liquid.
A.
Balancing the microcentrifuge. A microcentrifuge is meant for holding ‘microcentrifuge tubes’, which typi-
cally hold volumes between 0.5 mL and 2 mL. The Lysis Buffer, cells, and lysozyme in your tube of lysed cells
will amount to about 1.1 mL, so your tube is probably quite full.
Your centrifuge should always be balanced. This means you should place another tube of equal mass in the
open spot directly opposite from your Lysis Buffer tube. Inside the Plate Extract-it Kit, you will nd a Balancing
Tube which may already contain some water in it. If it does, note that while it will be close to the corresponding
mass you want, you should always double-check to make sure it is correct. To balance your tube, use a new
clean pipet to add tap water to the Balance Tube until the liquid level is similar. Use your small scale to weight
the tubes and conrm they weight the same.
The tubes should be within 5% mass of each other. For example, if Tube A = 2 g, then Tube B should be no more
than 2.1 g or less than 1.9 g because 5% = 0.05 x 2g = 0.1g. The closer the mass, the better. Make sure to close
the tubes back tightly after balancing them.
B.
With your experiment’s lysis tube set opposite your balance tube, close the microcentrifuge lid(s) as per the
manufacturer’s instructions, tighten the lid, set the speed at the highest setting, and start the microcentrifuge.
Monitor to see if the microcentrifuge vibrates - if it does, stop it immediately! Note that the microcentrifuge will
hum and make some noises as part of its normal operation. A vibration means you either did not place your
tubes opposite of each another, or you have not adequately balanced the tubes.
Pellet
Figure 5-8. Step 9. Pelleted cells in a microcentrifuge tube.
Centrifuge
Warning!
If this is your rst time using a microcentrifuge, set it up as per the manufacturer’s instructions. Then, start by spin-
ning the microcentrifuge without tubes in it. Try different speeds, starting slowly. As the rotor speeds up, there should
be no vibrations; it should be balanced. You should observe the same behavior when you add your balanced samples
into the microcentrifuge; they should not cause the microcentrifuge to vibrate.
If there is any vibration, immediately stop the microcentrifuge and re-balance your samples.
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128 Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Spin at maximum speed (13,000 x g - 15,000 x g) for 20 minutes. During this time, your proteins stay dissolved
in the liquid, while other macromolecules and cellular debris are forced to the bottom into a pellet.
At the end of this step, there should be a transparent liquid at the top that has your desired trait (e.g., for a purple
pigment, the liquid will be translucent purple) and, in the bottom of the tube, there should be a pellet of nearly
white cellular debris. There may be some trait (e.g., color pigment) remaining in the cellular debris and this
simply means that not all of your cells lysed. If this occurs, in future experiments you can let your cells incubate
longer in the Lysis Buffer and Lysis Accelerator.
Step 10. Extraction: Filter sterilize your proteins
Once you have lysed and pelleted the cell debris, there should be few remaining bacteria in the clear solution.
We’ll call this the sample. To be sure that there are no living bacteria or other microorganisms in the sample,
you will now lter sterilize it. This not only helps ensure safety, but it also helps ensure that your samples will
not spoil if stored for prolonged periods of time.
The lter provided in the Plate Extract-it Kit screws into the end of the supplied syringe to help press your
sample through the lter. Within the lter, there is a membrane with very small pores that allow molecules
like proteins, DNA and sugars through, but it traps larger molecules and entire bacteria. The lter provided
has pores that are 0.22 um in size. Can you remember how large a typical E. coli bacteria is? You can nd the
answer in Chapter 3.
Caution!
In the next step, be aware that if you apply too much pressure to the syringe, the syringe may ‘burst’ or the lter could
separate at the seam, resulting in a high-pressure stream of ying liquid. So do not press too hard while ltering your
solution. It is recommended that you wear safety goggles and a lab coat.
If you nd that it is getting increasingly harder for the liquid to pass through the lter, or has stopped passing through
entirely, it is likely that cell debris from the pellet has clogged the lter. If this is the case, you should acquire a new lter.
Figure 5-9. Step 10 -There may still be viable bacteria in the extract, so a lter is used to sterilize the sample.
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