86 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Chapter 4
Genetic Engineering Your E. coli Cells
You have now extracted DNA from fruit cells. You have
made LB agar plates. You have grown K12 E. coli cells
using two different methods: streaking them onto a
plate to get fresh bacteria paint and by painting them
to create living BioArt.
Consider your BioArt for a moment. Did it turn out
the way you expected? Painting with bacteria can
be challenging because the cells can grow beyond
where you’ve painted, distorting the image. As with all
science and art skills, practice makes perfect!
In this chapter you are going to use the skills, you
developed in Chapter 3 to take the next step on your
genetic engineering journey. You are going to streak
and grow cells in order to engineer them with DNA.
You will change the actual blueprints of the cells so
that they create something for you. This is the basis
of genetic engineering.
In practical terms, you are going to learn how to insert
DNA into cells and have the cells read and execute
the instructions encoded in the new DNA. This won’t
require you to replace the genome of the K12 bacte-
ria. Instead, you will be adding some additional DNA,
called a DNA plasmid, into the cells. Once inserted,
the bacteria will start to read and execute the DNA
from the plasmid, just as they will continue to read
and execute their normal genome “programming”.
The Fundamentals in this chapter will build on every-
thing you’ve learned in prior chapters. We will look
at how a cell reads DNA and begins ‘executing’ the
instructions to create something, a product, for
you. The process of reading a DNA plasmid is called
transcription, and it is one of the most important
processes in biology. All of this happens on the
“factory oor” in the cytoplasm of your K12 E. coli and
mostly involves nucleic acids and proteins.
Just as you were introduced to the concept of genes in
Chapter 1, in this chapter, you are going to learn rst-
hand how to insert genes into your K12 E. coli bacteria.
Once the gene is inserted via a plasmid, we will learn
how the cell reads the gene in order to start creating,
or microfacturing something for you. Scientists also
call this “expressing a trait”. “Microfacturing” is a
term that we use a lot throughout this book, but it is
rather new to the eld of genetic engineering! Since
you imagine the cell as a microscopic factory (micro-
factory), think of its activities as microfacturing.
Learning these subjects is the foundation of genetic
engineering and critical to becoming a Genetic Engi-
neering Hero. Once you understand how to insert DNA
into a cell and how a cell reads and executes a “DNA
program”, you’ll be on your way to learn advanced
subjects like designing, making and executing your
own “DNA programs.”
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87Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Getting Started
Equipment and Materials
The Amino Labs’ Engineer-it Kit
TM
is contained in the Zero to Genetic Engineering Hero Kit Pack Ch. 1-4 and
includes all the required pre-measured ingredients. The Keep-it Kit
TM
is optional and both can be ordered
separately at https://amino.bio/products.
Shopping List
Wetware kit: Amino Labs Engineer-it Kit
TM
(https://amino.bio)
Wetware kit: Amino Labs Keep-it Kit
TM
(https://amino.bio) (optional)
Minilab (DNA Playground)
Microwave
Laptop/Desktop PC for Virtual Bioengineer
TM
Engineer-it Kit Edition
(https://amino.bio/pages/vbioengineer)
Instructions Overview
Day 1
1. Practice Engineering E. coli by completing the entire Virtual Bioengineer
TM
simulation
2. Make non-selective and selective LB agar plates
3.
Streak E. coli bacteria on non-selective plates, and incubate in your DNA Playground for 12-24 hours
Day 2
4. Make chemically competent cells so your E. coli can take in the DNA plasmid
5. Heat shock and transform your DNA & E. coli
6. Recover your cells with enriched growth media
Day 3
7. Plate your transformed & recovered cells on selective LB agar plates
8. Incubate your plates for 24-48 hours
Day 4
9. View results
Day 5
10. Optional: Immortalize your rst genetic engineering experiment (Keep-it Kit
TM
)
Chapter Timeline Overview
Timeline to complete the hands-on exercise is:
Day 1: ~60 minutes followed by 12-24 hours incubation,
Day 2: ~60-90 minutes followed by 12-24 hours recovery
Day 3: ~20-30 minutes followed by 24-48 hours incubation
Day 4: ~15 minutes to view results. Optional: Prepare for preservation followed by 24 hours of drying time
Day 5: Optional: ~15 minutes to immortalize results followed by 24 hours of curing time.
Timeline to read Fundamentals is typically 3 hours.
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88 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Virtual Bioengineer
TM
Interactive Sim Practice Breakout Session 1
In Chapter 3, you completed Virtual Bioengineer
TM
Canvas Kit Edition and learned how to make LB agar
plates, and practiced streaking/incubating K12 E. coli cells.
In this chapter, you will complete a different Virtual Bioengineer
TM
simulator, the Engineer-it Kit Edition,
and also do those steps in real life. The simulation will familiarize you with the materials and procedure
that you will be using to:
• Make selective and non-selective LB agar plates
• Streak cells and incubate them
• Collect colonies to make competent cells
• Add DNA and heatshock the cells to get your DNA into the cells
• Recover the cells
• Incubate the cells
Virtual Bioengineer
TM
is free and takes about 20-30 minutes to complete. It is recommended that you
complete the simulation a few times. That way, when you do the hands-on exercises with real DNA and
cells, you won’t make any mistakes!
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89Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Learning Hands-On: Transforming K12 E. coli cells with
a DNA Plasmid
Now that you have a good understanding of DNA, proteins,
and the structure of E. coli cells, it’s time to complete your rst
genetic engineering exercise! You will insert circular strands of
DNA, called DNA plasmids into cells. The DNA plasmids contain
genes that genetic engineers have designed to make your cells
resistant to a common laboratory antibiotic and to produce a
colored pigment.
You will build on your mastery from Chapter 3 by growing E.
coli cells. Then, using chemical processes, you will insert DNA
plasmids into your cells so that they read and execute the genes
in the DNA plasmid. This will produce the pigment specied by
the plasmid.
Step 1. Download the instruction manual for the Engineer-it Kit
Familiarize yourself with the Engineer-it Kit instructions found at https://amino.bio/instructions. These may
reect updates to the kit’s material and instructions. The instructions will also specify what to look for when
completing each step. If there are any major conicts between the most recent manufacturers’ instructions
and this book, use the instructions as your primary resource.
Step 2. Put on your gloves and lab coat
Remember to always put on your gloves and lab coat!
Figure 4-1. Step 1 Download the Engineer-it Kit instructions.
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90 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Step 3. Label your plates
Get your Engineer-it Kit from the refrigerator and set up your DNA Playground. Take the four plates from the
Engineer-it Kit and use a permanent marker to label the bottom of each plate as follows:
1. NS; [your initials] 3. S; [your initials]; (E)
2. S; [your initials]; (+) 4. S; [your initials]; (-)
These abbreviation stand for : NS = non-selective, S = selective, (E) = experimental sample, (+) = positive control
sample, (-) = negative control sample
Step 4. Make non-selective and selective LB agar plates
Following the manufacturer’s instructions, make LB agar plates to grow non-genetically engineered bacteria as
well as genetically-engineered bacteria. For this, you will create two kinds of plates: one plate without antibi-
otics and three plates with antibiotics. Complete the exercise according to the kit instructions and techniques
you learned in Chapter 3.
This will be similar to making plates for Chapter 3’s Canvas Kit, except this time you will leave one plate without
antibiotics. This will be your non-selective plate (NS), which you will use on Day 1, along with your selective S(-)
plate. Return the other two selective S(E) and S(+) plates to their resealable bag in and store in a refrigerator
until Day 3.
Selective and Non-selective Going Deeper 4-1
Non-selective LB agar plate: This growth plate contains only LB agar, NO antibiotics. They are used to
grow bacteria that have not yet been genetically engineered with a selection trait, for example, antibiotic
resistance. The K12 E. coli bacteria that you grew in Chapter 3 were already engineered with a DNA plasmid
to make them colorful and resistant to the antibiotics. This is why in Chapter 3 all of your LB agar plates
had antibiotics.
Selective LB agar plates: These plates contain the same LB agar that bacteria need to grow and divide,
with something extra added while the agar is still hot and molten. This addition is usually an antibiotic
such as ampicillin, carbenicillin, chloramphenicol, kanamycin, or tetracycline. When you engineer your
bacteria with the plasmid, the plasmid contains a ‘selection marker’ – a gene – that results in the creation
of a protein that can break down antibiotics. This is called antibiotics resistance, and it makes the bacteria
“immune” to the antibiotic. As you will see further in this chapter, this helps you to select for bacteria that
you’ve engineered.
Figure 4-2. Step 4 Prepare your non-selective and selective plates.
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