Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 51
Chapter 3
Growing E. coli Cells
In Chapter 1, you learned about the structure of DNA
and how it is a microscopic chemical string of nucle-
otides that is the blueprint for living cells. Cells know
how to read DNA to build their structural components
and internal machinery, as well as to create enough
energy to grow and replicate.
In this chapter, we will focus on Escherichia coli (E.
coli). E. coli is the microorganism that you will be engi-
neering in the experiments in this book. Microorgan-
isms are organisms that are not visible to the naked
eye, like bacteria, viruses, and some fungi. E. coli is
a type of bacteria from the Escherichia family. There
are many types of E. coli, and all are living cells that
contain a DNA blueprint.
The E. coli you will be using is not to be confused
with the E. coli you may hear about in the news that
can cause illnesses in humans or animals. The E. coli
bacteria used in the Genetic Engineering Hero’s jour-
ney comes from a safe lab strain of E. coli called K12.
It is the most widely used organism in life science
research labs around the world.
In this chapter, you will learn the skills and princi-
ples behind growing E. coli in a petri dish, an essen-
tial skill to master on your journey to becoming a
Genetic Engineering Hero. You will make the “food”
and surface (substrate) that E. coli grow on. You will
learn about the environmental conditions that are
required to grow E. coli. You will learn the necessary
techniques to grow cells so that they can be useful for
other experiments.
Like Chapter 1, the Going Deeper sections provide
context and more in-depth knowledge. They will give
you a deeper understanding of what is happening
throughout your experiment so that you can master
the techniques and even modify them in future exper-
iments. If this is your rst time growing bacteria, you
may want to repeat the exercise twice: Once with a
focus on the hands-on instructions, and a second time
focussing on the Going Deeper sections.
In the Fundamentals section, we will examine the
“cells-as-factories” analogy, learning more about
bacteria in general. In Chapter 1, we learned about
the DNA as blueprints. In this chapter, we will take a
tour of the entire E. coli microfactory, visiting the ve
key components of an E. coli cell:
• Capsule layer: outer protective shield of E. coli
•
Outer membrane: primary outer structural barrier
•
Intermembrane space: a key passageway to enter
and exit the cell
• Inner membrane: an inner structural barrier
•
Cytoplasm: the primary space in which the cellular
activities occur
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 52
Keep in mind!
The E. coli you will use throughout this book is a specic lab strain that is safe. In fact, most types of E. coli are not
infectious and are actually necessary for your digestive system to function.
E. coli have evolved over millions of
years inside the large intestine of animals such as humans. By colonizing our large intestines, they help us digest
food, create vitamins, and produce amino acids for us. One strain of
E. coli is a widely used probiotic that helps to
reduce bloating in the intestine.
The strain of E. coli included in the Amino Labs’ kits was rst collected by scientists in the early 1900s. It has been
used in thousands of labs worldwide and in countless experiments for nearly 100 years, with no reported incidences
of harm. The E. coli within the kit also has further deciencies, making it unlikely for it to survive outside your
petri dish. You will learn more about this later on in the chapter. For now, remember that this bacteria is safe to
use in your experiments. Even so, to get used to standard science safety procedures, you must always follow safety
practices you learned in Chapter 2, no matter how safe the type of bacteria.
Figure 3-0. Low-temperature electron micrograph of a cluster of E. coli bacteria, magnied 10,000 times. Each individual bac-
terium is oblong shaped. 2005. Photo by Eric Erbe, digital colorization by Christopher Pooley, both of USDA, ARS, EMU., Public
domain, via Wikimedia Commons
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 53
Getting Started
Equipment and Materials
For this chapter, you will need a Canvas Kit
TM
from Amino Labs. The Amino Labs’ Canvas Kit
TM
is part of the
Zero to Genetic Engineering Hero Kit Pack Ch. 1-4 and includes all the required pre-measured ingredients. This
kit can also be ordered separately at https://amino.bio/products.
You will also need a standard box of Jell-O
TM
for the Practice Exercise. The equipment you will need includes a
DNA Playground Minilab, a microwave, and a computer (not a tablet or phone) to complete an online simulation.
Shopping List
Pre-practice exercise:
Box of Jell-O
TM
or Jelly
TM
Main exercise:
Wetware kit: Amino Labs Canvas Kit
TM
(https://amino.bio)
Wetware kit (optional): Keep-it Kit
TM
(https://amino.bio)
Minilab (DNA Playground)
Microwave
Laptop or Desktop PC with internet connection for completing Virtual Bioengineer
TM
Canvas Kit Edition.
Instructional Overview
A. Pre-practice by making Jell-O
TM
B. Pre-practice simulation using Virtual Bioengineer (https://amino.bio/vbioengineer)
1. Download the manufacturers Canvas Kit
TM
instructions from https://amino.bio/instuctions
2. Put on your gloves and lab coat
3. Creating molten LB agar
4. Adding antibiotics
5. Pouring LB agar plates
6. Use & storage of LB agar plates
7. Streaking and painting E. coli bacteria
8. Incubating E. coli cells
9. Viewing plates of grown bacteria
10. Preserving your bioart (optional - using Keep-it Kit
TM
)
11. Clean up and inactivation
Chapter Timeline Overview
Timeline to complete the hands-on exercise is:
Day 1: ~20 minutes Breakout Session 1, ~20 minutes Breakout Session 2,
Day 2: ~60 minutes to make LB agar petri dish and streak cells, followed by 24-48 hours incubation
Day 3: ~30 minutes to paint your living paintings followed by 24-48 hours incubation
Day 4+: ~15 minutes to view results.
Timeline to read Fundamentals is typically 3 hours.
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 54
Making Jell-O
TM
Breakout Session 1
One of the rst lab skills necessary to master in genetic engineering is the creation of LB agar plates. LB
agar is a substance a bit like Jell-O
TM
. You pour the agar as a hot liquid into a container – in this case, a
petri dish (also known as a plate). The agar then cools to become the gelatinous substrate (or surface) on
which the E. coli cells feed and grow. Since the principles of making LB agar plates are similar to making
Jell-O
TM
, that’s where we’ll start.
When making Jell-O
TM
, you mix the powder with boiling water. It is important that this water is boiling
because otherwise, the gelatin (gelling agent) in the Jell-O
TM
will not dissolve. Once everything is mixed
together, the solution becomes transparent, with no particles are oating around.
The Jell-O
TM
instructions suggest you add cold water to accelerate the cooling process. You will not do this
when making your LB agar plates but do follow your Jell-O
TM
instructions. Then, you will pour your Jell-
O
TM
mix into a container and let the solution cool down and solidify. At the end, you have a transparent yet
colorful gel. This is very similar to LB agar plates. Since you will be spreading (streaking) cells onto the
surface of the agar in the main experiment, try running a spoon or chopstick on the surface of the solidi-
ed Jell-O
TM
to get acquainted with its texture and softness. This will help you work well with agar later on.
The key differences between Jell-O
TM
and agar are:
•
Jell-O
TM
uses gelatin, a protein-based gelling agent from animals, whereas agar uses a sugar-based
gelling agent from algae.
• LB agar has sugar, nutrients, amino acids, and minerals for bacterial growth - while Jell-O
TM
, for the
most part, has only sugar. Yum!
• Jell-O
TM
includes food dye to change its color, while LB agar is naturally a pale yellow due to the vita-
mins and other cellular nutrients.
•
Jell-O
TM
requires you to add cold water and refrigerate it to speed up the “gelling” process. With LB
agar plates, you do not have to do this because:
a) the agar gels pretty quickly (10-15 minutes)
b) it could compromise the sterility of your agar (by contaminating them with organisms in tap water).
c) the antimicrobial chlorine and/or uoride in tap water would prevent the growth of your bacteria.
Virtual Bioengineer
TM
simulation Breakout Session 2
The Virtual Bioengineer
TM
Canvas Kit Edition is an online sim-
ulation that mimics the procedure you will use to make LB agar
plates. As you will see, making LB agar plates applies similar
principles to making Jell-O
TM
. However, the procedure and ma-
terials are different.
Virtual Bioengineer
TM
Canvas Kit Edition will familiarize you
with the materials and procedure used in this chapter. The sim-
ulation is free and will take less than 20 minutes to complete.
https://amino.bio/vbioengineer
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Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 55
Learning Hands-On: Growing K12 E. coli cells
Step 1. Download the instruction manual for the Canvas Kit
Familiarize yourself with the Canvas Kit instructions found at https://amino.bio/instructions.
The manufacturer’s instruction manual has the most up to date procedures for this exercise and may be slightly
different from this book. If there are any differences between this book and the up-to-date manufacturers’
instructions, always follow the manufacturer’s instructions.
Step 2. Put on your gloves and lab coat
When doing genetic engineering and science experiments, you should practice what is called aseptic technique.
Aseptic technique is the use of methods and procedures to prevent cross-contamination of microorganisms
between your experiments, your environment, and you. Much of the following has been talked about in Chapter
2, but let’s quickly refresh the topic.
You have lots of microorganisms on your hands that can easily be transferred into the petri dishes you will be
making in this exercise, and can result in contamination of your experiment. The same goes for your clothing,
mold in your environment, your pets, and even your hair. Conversely, you will be growing microorganisms
throughout this book, and you want to prevent them from getting onto your skin and body.
Practices like wearing nitrile or latex safety gloves at all times during experiments is a great way to prevent the
transmission of microorganisms in either direction. Wearing a lab coat will also help prevent the transfer of
microorganisms between the rest of your body and your experiments. If you have long hair, you should tie it up
to prevent it from making contact with your experiments. While it is always recommended that safety glasses
be worn, you will rarely see this as common practice in research labs. If you have a respiratory infection, you
can wear a mask to prevent any microorganisms in your breath from getting in your experiments. Make sure to
never touch your face with gloved hands. If you do, wash your face with soap and water and change your gloves.
It is also good practice to put the inactivation bag from your Canvas Kit into a discard container now so that it
is ready for use and won’t tip over.
Figure 3-1. Step 1 Download the Canvas Kit instruction manual. Make sure to choose the right one from ‘Individual or Classroom’
based on the kit you have.
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