Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 66
on completing your second experiment!
Congratulations on growing E. coli cells on LB agar plates that you made yourself. This is the rst major step
toward becoming a Genetic Engineering Hero.
The hands-on exercise was packed with new information and essential skills. In the following section, you will
take a deeper look at the fundamentals of E. coli. We will...
• Look at the history of the K12 E. coli cells you grew.
• Examine the structure of E. coli: what they look like, what they’re made of, what helps them grow.
•
Just as you learned about the DNA macromolecule in Chapter 1 Fundamentals, we will look at three other
important macromolecules: sugars, proteins, and lipids.
This section features a tour of a K12 E. coli ‘microfactory’ which in many ways is like a traditional factory that
you might see in your everyday life.
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 67
Fundamentals: E. coli Cells
Introduction to “Lab” E. coli
Escherichia coli (E. coli) is generally associated with
hamburger meat and infection. However, there are
many different strains (types) of E. coli, and most
are not pathogenic (Figure 3-13). In fact, E. coli aids
in digestion and helps to produce amino acids and
vitamins for our bodies. Although we don’t have the
evidence yet, it is possible that every human has at
least some E. coli bacteria as part of their large intes-
tine microora.
E. coli is the most widely used organism in scientic
research, and is one of the simplest organisms that
can be used in genetic engineering. If you are famil
-
iar with using software, you can think of E. coli as a
“Lite” version of a software suite. Compared to other
organisms like yeast cells or mammal cells, which
have many more cellular “bells and whistles”, E. coli
is simple to engineer, grows fast (divides every 30
minutes), and is reliable (Figure 3-14).
E. coli was rst observed in 1885 by the German-Aus-
trian pediatrician Theodor Escherich, after whom
the bacteria is named. E. coli may have a prevalence
of around 0.1% of “normal gut ora”. There are more
than 700 strains of E. coli, including the lab strain you
will use throughout this book. Only a few versions of
E. coli, such as strain O157:H7, are pathogenic due to
the production of bacterial toxins. These pathogenic
strains of E. coli are Risk Group 2 (RG-2) organisms and
should only be explored using Containment Level 2
Laboratory guidelines and conditions, which requires
government approval.
On the opposite end of the spectrum, the E. coli strain
DSM 6601 (also known as Nissile 1917), was isolated in
1917 from a World War I soldier who had a resistance
to diarrhea, and who was known for having a “strong
stomach”. E. coli DSM 6601 has now been used as a
probiotic for over 100 years!
The strain you will be using throughout this book is
called the “K12” strain of E. coli - this E. coli is proba-
bly the most studied and well-understood organism…
ever. Originally isolated at Stanford University in the
Figure 3-13. There are many different ‘versions’ of E. coli.
Scientists are responsible for making some to better un-
derstand how E. coli functions, but many have also naturally
evolved to be different.
E. coli (bacteria)
P. pastoris (yeast)
Free floating genome
Flagella
Cell membrane
Mitochondria
Nucleus enclosed genome
Peroxiomes
Vacuole
Golgi apparatus
Cell membrane
Prokaryotic
Eukaryotic
Figure 3-14. Bacteria like E. coli are simple cells with free-oating genomic DNA, cell membranes and agella. Yeast cells are much
more complex and have other specialized cell structure/compartments that the cells use to produce energy, make or break down
fats, a nucleus to store DNA and a golgi apparatus to help fold and package products and machinery that the cells produce.
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 68
1920s, the K12 strain has gone through much evolu-
tion and manipulation in labs to become the lab strain
we take for granted today.
Recall learning about plasmids at the end of Chapter
1; the short circular DNA helix loops that are akin to
a USB stick for cells. In addition to a 4,600,000 nucle-
otide “main” chromosome, natural E. coli have a large
plasmid called an “F plasmid” (short for “Fertility
factor plasmid”). This plasmid enables one bacte-
ria to share DNA with another through a process
called bacterial conjugation - it allows the bacteria to
communicate with one another. In lab strains of K12
E. coli, these F plasmids have been removed by scien-
tists, making it very difcult for the bacteria to share
genetic information. This substantially reduces the
risk of K12 bacteria sharing information with other
organisms if they are released into the environment.
It also prevents communication between organisms
in your samples during your experiments. K12 E. coli
is not only a scientically-sound organism choice, it
is also a responsible one.
As you will learn in coming chapters, genetic engi-
neers sometimes use antibiotic resistance genes
during the genetic engineering process. A question
that sometimes arises in public discussion is whether
the antibiotic resistance genes used in genetic engi-
neering cause the emergence of “superbugs” resistant
to all antibiotics. The short answer is no, and one of
the main reasons for this is that lab strains of E. coli
have been “gagged” by the engineers - they cannot
really share their DNA with other cells thanks to the
removal of the “F plasmid”.
A second signicant difference between Lab K12 E. coli
and other forms of E. coli is that they no longer have a
“lambda phage” infection. Lambda phage is a virus
that infects E. coli and becomes part of its genome
(Figure 3-15). Yes, even bacteria can get viruses and
catch a cold! The original K12 strain has the lambda
phage in its genome. Under the right conditions, the
virus can become active, kill the cells and spread to
others. Lab strains of K12 E. coli no longer have this
infection, and you do not have to worry about your
bacteria catching a cold during your experiments.
Throughout the 1950s-1980s, experiments were
completed to rst remove lambda phage and then the
F plasmid, as well as a few other genes to achieve a
version of K12 E. coli called “MG1655”. Further tweak-
ing to this bacteria has led to the most widely used
E. coli K12 bacteria strains, called DH5α and DH10β.
Labs have made hundreds of other versions of E. coli
to study more about how cells work, but the DH5α and
DH10β versions remain the most commonly used in
genetic engineering. These are the E. coli strains you
will be using and encounter in this book. For now, the
K12 E. coli that you have already used in the hands-on
exercise and whose ancestor was originally isolated
from the feces of someone at Stanford University
in California, about 100 years ago will be the model
organisms we study.
Figure 3-15. The lambda phage is a virus that can infect E. coli.
Microflora Going Deeper 3-4
Microora is the name given to all of the microorganisms in and on the human body that coexists with you
and, in most cases, keep you healthy. Microora may also be called ‘normal ora’ or your ‘microbiome.’ E.
coli is just one of the hundreds of different kinds of bacteria that live on your skin, in your armpits, eyes,
mouth, intestines, and even on your hair. In the 19th and 20th centuries, the consensus was that most, if not
all, bacteria were bad. However, in the 21st century, we now know that all the bacteria in our microbiome
(E. coli included) are essential for our health.
If you’re interested in learning about your microbiome, you can use one of the many consumer microbiome
testing services available online for non-medical purposes. Typically, the testing company will send you a
swabbing kit and, after swabbing your body, you send it back for analysis using next-generation genomic
sequencing. Once analyzed by the company, you can then go to your online account to view the results. Fun!
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 69
Fence line
Brick wall
Lobby
Inner wall
Main manufacturing floor
Machinery, computers
& workers
manufacture
E
E
Capsule layer
Outer membrane
Intermembrane space
with peptidoglycan
Inner membrane
Cytoplasm
main microfacturing area
DNA & proteins
microfacture
E
E
Figure 3-16. Factory and microfactory tour map. The tour will move from fence line to manufacturing oor; capsule layer to cyto-
plasm.
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 70
A Tour of the E. coli Microfactory
Now that you’re familiar with the history and origin of
the K12 E. coli that you streaked and painted on your
LB agar plates, the rest of the Fundamentals involve
digging deeper into what makes E. coli tick. Let’s have
a look “under the hood”!
In Chapter 1, we learned what makes up the macro-
molecule DNA: CHOPN. We saw that CHOPNS (carbon,
hydrogen, oxygen, phosphorous, nitrogen, sulfur)
have been classified as organic elements because
they are commonly found in organisms. You can refer
to the periodic table at the end of the book to refresh
your memory. In this chapter, you’ll see that all other
macromolecules like sugars, proteins, and lipids also
include CHOPNS in their make-up.
To make this information more relatable, we are going
to use a cells-as-factory analogy, starting with a tour
of the E. coli ‘microfactory ’. We call it a microfactory
because when E. coli is used in genetic engineering,
the cells literally become a factory. They read instruc-
tions (the DNA you insert into the bacteria), take in raw
materials (the nutrients from the LB Agar), and create
a product (like insulin from the Industry Breakout: Insu-
lin Going Deeper 3-5).
The goal of this chapter is to understand the overall
structure of an E. coli cell and the atoms, molecules,
and macromolecules that it is made of. We’ll look at
ve key parts of the microfactories (Figure 3-16):
•
The capsule layer (A): the outermost protective
shield of E. coli.
•
The outer membrane (B): the primary exterior
structural barrier.
•
The intermembrane space (C): a key passage for
entering and exiting the cell.
•
The inner membrane (D): a structural barrier
within the cell.
•
The cytoplasm (E): the primary space in which
cellular activities occur.
The Fence (A)
The outermost property line of a factory is defined
by a fence. This forms the outermost barrier of the
factory, protecting the entire property from outside
Industry Breakout: Insulin Going Deeper 3-5
Insulin is a protein made in the pancreas and exists within the blood to regulate the amount of sugar
that cells can absorb. When a person cannot make insulin in sufcient quantities, they have type I diabe-
tes. Treatment of type I diabetes began in the 1920s after the discovery of insulin. Back then, insulin
was extracted from barnyard animals by harvesting and grinding up their pancreases. Insulin was then
extracted, puried and injected into humans with diabetes. While this form of insulin did treat diabetes, it
had undesirable side effects, including allergic reactions, and required a lot of animal pancreases.
Visit https://amino.bio/insulin to be redirected to a website with a history of insulin. Here, you will nd an
image of a massive pile of pig pancreas that were used to produce only 8 ounces of insulin.
In the 1970s, the fathers of modern biotechnology (scientists Stanley N. Cohen and Herbert Boyer) devel-
oped methods to ‘cut and paste’ DNA from one organism to another. In 1976, Boyer founded a company
called Genentech. Their rst project was nding the DNA blueprint for human insulin so it could be inserted
into E. coli bacteria. They were successful and created the rst modern biotechnology product: human
insulin. In 1982, humanity ushered in a new era of manufacturing - using bacterial cells as microfactories
to deliberately produce important end products for a commercial purpose. It is estimated that over 100
million people rely on microorganism-produced insulin every year to stay healthy and alive. Now, hundreds
of other medicines are produced using genetic engineering and biomanufacturing methods, both of which
you will become familiar with in later chapters.
Figure 3-17. The outer fence or capsule layer made of lipo-
polysaccharides (LPS) protect the factories and are anchored
in place.
Sugar
Chain
Lipid
Anchor
Post and
Chain Mesh
Concrete
Anchor
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