119Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Chapter 5
Extracting your engineered proteins
In Chapter 4 you made a monumental leap in ofcially
becoming a genetic engineer! Congratulations!
By making LB agar plates, growing cells, making those
cells chemically competent, then heat shocki
ng, recov-
ering and incubating them, you were able to insert
DNA plasmids into some blank K12 E. coli cells. While
your cells were recovering, the ones that took up a
DNA plasmid immediately began the Three Steps to
Microfacturing. We focused on the rst step called tran-
scription where DNA is read and transcribed by RNA
Polymerase into a different nucleic acid, RNA.
The cells also simultaneously started the second
of the Three Steps to Microfacturing, a process called
translation. That’s the primary focus of this chap-
ter. Translation involves reading an RNA transcript
and translating it into a chain of amino acids, called
proteins. As mentioned in Chapter 3, proteins are
usually the ‘machinery’ of cells, and in Chapter 4’s
hands-on exercise, you manufactured two different
kinds of proteins: i) the protein color pigment; ii) the
protein enzyme that enables the cells to be resistant
to the antibiotic in the selective plates.
These are two examples of an unimaginable number
of different proteins – each with a different function
– being microfactured at any given moment across
millions of species and organisms on planet earth.
The goal of this chapter’s hands-on exercise is to
combine and extend on the principles that you learned
in Chapter 1 (lysing cells), Chapter 3 (growing cells),
and Chapter 4 (engineering cells). This chapter will
help you learn how to amplify and extract the proteins
that you’ve engineered the cells to microfacture in
order to start using them outside of the cells. Your nal
result will be a tube of ‘cell extract’ containing a large
quantity of your microfactured product, such as your
colored proteins.
Using your Minilab and microcentrifuge, you will:
•
Grow and engineer cells with a DNA plasmid to
make freshly engineered cells.
• Create several more selective LB agar plates.
•
Streak the selective LB agar plates with your engi-
neered cells to amplify them.
• Incubate your plates to allow for the growth of cells
and expression of the proteins.
•
Collect the cells and lyse them to extract your
microfactured proteins.
•
Centrifuge the samples to ‘pellet’ other ‘cellular
debris’.
•
Sterilize the extract to remove any remaining
bacteria.
In the Fundamentals section, we are going to dive into
the second step of the Three Steps to Microfacturing -
Translation. You will notice many similarities between
how transcription and translation work. While differ-
ent protein enzymes are involved, you will begin to see
some general themes start to emerge. As these themes
start to become clear in your mind, you will know that
you are beginning to understand how cells operate
and how to engineer them!
As with transcription in Chapter 4, we will cover: How
cells know how to start translation, do translation, and
stop translation. With a solid grasp of these funda-
mentals, you will know the basics behind the Three
Steps to Microfacturing, and begin to understand the
underlying chemistry behind genetic engineering.
With this, you will be ready to take on the nal step of
microfacturing in Chapter 6, Enzymatic Processing,
where you will also learn about atoms and
bonding.
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120 Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Getting Started
Equipment and Materials
The Amino Labs’ Engineer-it Kit
TM
and Plate Extract-it Kit
TM
include all the required pre-measured ingredi-
ents. These kits be ordered separately at https://amino.bio/products
Shopping List
Amino Labs Engineer-it Kit
TM
(Optional) For this experiment, you can use your saved Engineer-it Kit
S(e) or S(+) petri dish, refrigerated for no more than 1 week. Use a new Engineer-it Kit to get freshly
engineered cells, or use the tube of pre-engineered cells that comes with the Plate Extract-it Kit
TM
.
Amino Labs Plate Extract-it Kit
TM
(https://amino.bio)
Minilab (DNA Playground)
Microwave
Microcentrifuge
Instructions Overview
Prepare: Get Freshly Engineered cells
1. Use your freshly engineered cells from your refrigerated S(e) plate (if it is no more than 1 week old),
repeat the Engineer-it Kit exercise from Chapter 4 to obtain fresh colonies of engineered bacteria,
or use the pre-engineered cells that come in the Plate Extract-it Kit.
Day 1: Amplify your Engineered cells
2. Use the Plate Extract-it Kit to create selective LB agar plates. Pick fresh colonies to spread onto two
selective LB agar plates.
3. Incubate the petri dishes for 24-48 hours until the streaked cells grow and express the desired trait
(e.g., color pigment).
Day 2: Extract proteins
4. Collect the cells by using an inoculating loop to scrape the cells from the surface of the selective LB
agar petri dishes and deposit them into a tube of Lysis Buffer.
5. Lyse the cells by using Lysis Buffer and enzymes from the Extract-it Kit to break the cells open and
release the microfactured products.
6. Incubate lysing cells at room temperature (color proteins) for 1 to 24 hours (or up to 72 hours if you
incubate in the refrigerator).
Day 3: Sterilize proteins
7.
Centrifuge the sample using a microcentrifuge to ‘spin’ the samples at max speed (13,000 x g or higher)
to pellet the micelles and cell debris while leaving the extracted proteins dissolved in the liquid.
8. Filter s
terilize the sample as there may still be viable bacteria in the extract.
Chapter Timeline Overview
Timeline to complete the hands-on exercise is:
Preparation: If you are completing a new Engineer-it Kit, this will add 3 or 4 days of hands-on exercise
Day 1: ~60 minutes followed by 24-48 hours incubation
Day 2: ~30 minutes followed by 1-72 hours incubation
Day 3: ~60 minutes
Timeline to read Fundamentals is typically 3 hours.
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121Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Learning Hands-On: Culture and lyse engineered E. coli
to obtain a protein product extract
Step 1. Download the instruction manual for the Plate Extract-it Kit
Familiarize yourself with the Plate Extract-it Kit instructions found at https://amino.bio/instructions. These
may reect updates to the kit’s material and instructions and also include what to look out for when completing
the steps. If any conict between these and the manufacturers’ instructions happen, always follow the manu-
facturer’s instructions.
You may also need the Engineer-it Kit instructions if you are doing the genetic engineering of bacteria again.
Step 2. Put on your gloves and lab coat
Step 3. Transform cells to get fresh colonies (Optional)
For this exercise it is best to have fresh colonies of genetically engineered E. coli cells on selective plates. If
you have completed the exercise in Chapter 4 in the last week and your selective LB agar plates with engineered
bacteria were stored in a way that they did not dry out (in a ziplock-style bag in a refrigerator), you can use a
bacterial colony from one of those plates. If you have a new Engineer-it Kit, you can repeat the Engineer-it exper-
iment. Practice makes perfect! By repeating the exercise, you will rene your transformation skills, which are
amongst the most important for genetic engineers. Alternatively, you can use the pre-engineered cells that come
with the Plate Extract-it Kit being aware that your best results would come from freshly engineered colonies.
When amplifying cells, it is always wise to use cells that have been freshly engineered. While this is not manda-
tory, it is good practice since the number of viable cells may be much lower on a plate that is not fresh. The
longer you store your cells, the higher the chance that they have died. Further, the longer you store your plates,
the higher the chance of seeing contamination. If you see anything that doesn’t look like your engineered cells
growing in your plates, this could be contamination and it is not recommended that you use any of the cells in
that petri dish, even if they appear “clean” and separate from the contamination. If it is a fuzzy mold growing,
spores could be throughout the plate and can contaminate plates that you’re trying to culture and spread to
other experiments!
Figure 5-1. Step 1. Download the Plate Extract-it Kit instructions. You may also need the Engineer-it Kit Instructions.
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122 Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
Step 4. Make selective LB agar plates for amplication
Once you have your engineered cells, you must now amplify them. To amplify your cells, you will use selective
LB agar plates made with your Plate Extract-it Kit. You will not need any non-selective plates for this exper-
iment, so you can add the antibiotics immediately after heating and entirely dissolving your LB agar powder.
In the prior chapters, you labeled your petri dishes with an “S” for the antibiotic-containing selective plates.
In this exercise, you can move one level closer to being a Genetic Engineering Hero by labeling the bottom
your petri dishes with the antibiotic abbreviation instead of “S”. Check within your Plate Extract-it Kit to see
which antibiotic is used. In Chapter 3, you learned about labeling your plates with a colored streak to identify
the antibiotic (Table 3-1). Another way to label plates is with an abbreviation for the antibiotics, as you saw in
Table 4-1. For example, if you are using the antibiotic chloramphenicol, you would label your plate with the
abbreviation “C” or “Chlor”. As you become more sophisticated in your experimentation, it is common to use
many different antibiotics to select for different engineered organisms. By labeling the specic antibiotic name
or abbreviation on the bottom of the plate, you will make sure you are using the correct selective plates for the
corresponding experiment.
Step 5. Culturing: Spread out your freshly engineered cells
Culturing is a general term for amplifying your cells using petri dishes or in liquid broth culture. Here you will
be spreading your engineered cells across two of the four selective plates to amplify them. The two remaining
plates can be refrigerated for your own experimentation.
If you are using cells from the S(e) or S(+) plate, select a fresh colony of engineered cells that is expressing the
Figure 5-2. Step 4. Prepare your Selective Plates from the Extract-it Kit to amplify the quantity of cells for extraction.
Figure 5-3. Step 5. Amplify your engineered cells by streaking them on two selective plates in a double zig zag pattern.
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123Zero to Genetic Engineering Hero - Chapter 5 - Extracting your engineered proteins
24h
deepest coloration and, using a yellow inoculating loop from the Plate Extract-it Kit, press the end of the loop
into this single colony to transfer it to the loop so that you may spread it on your new selective plates. If you are
using the tube of pre-engineered cells included in the Plate Extract-it Kit, dip your yellow inoculating loop in
the tube to tranfer the cells, just like you did in Chapter 3.
Spread the cells onto the rst selective LB agar plates by tracing a ‘dual zig zag pattern’ with your loop (Figure
5-3). Use the same loop for the entire spreading procedure as the goal of this exercise is to get lots of cells, like
you did for your bacteria paint in Chapter 3. Repeat this again on a second selective LB agar plate using the
same loop, but after selecting another colorful colony from the original plate, or dipping in the tube of pre-en-
gineered cells again. At the end of this step, you should have two selective LB agar plates that have engineered
bacteria spread across them.
Step 6. Culturing: Incubate at 37 ˚C for 24-48 hours
Just like in Chapters 3 and 4, it is recommended that you incubate the culturing cells at their optimal tempera-
ture, 37˚C for 24-48 hours.
24h
Figure 5-4. Step 6. Incubate your petri dishes at
37˚C for 24-48 hours until you see your bacteria colonies grow and be colorful.
Lysing cells: ‘more doesn’t mean better’ Pro-tip
The Plate Extract-it Kit has enough lysis buffer to support two plates of cultured bacteria. If you add cells
from more than two plates, you will not have enough surfactant to lyse all of the cells, and the experiment
will not work as well.
This is an excellent example of the classic science rule; “more does not mean better”. In fact, in scientic
research and genetic engineering projects, you have to use optimal experimental conditions. If you were
to use four plates of cultured cells and add them to the tube of lysis buffer from the Plate Extract-it Kit,
you would get less ‘cell lysis’ than by adding just two plates of cultured cells. This is because there won’t be
enough surfactant molecules (Triton X-100) to cut through the membranes of the extra cells effectively. In
trying to cut into twice as many cells, the molecules will be unable to lyse all the bacteria fully.
Imagine you have enough maple syrup to make 2 pancakes delicious. If you were to add 2 more pancakes
to the stack without adding more syrup, the 4 pancakes would not taste that great because there is not
enough maple syrup per pancake. Just as there is an optimal syrup to pancake ratio for each human, there
is an optimal surfactant to cell ratio.
Keep in mind!
The ideal temperature for E. coli growth is 37˚C. However, some proteins are more stable at lower temperatures so
30˚C or even room temperature can be used in some instances. If you like, you can incubate at 37˚C the entire time,
at 30˚C the entire time, or a combination of both such as 37˚C for 24 hours and 30˚C for another 24 hours .
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