147Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Chapter 6
Processing Enzymes
In Chapters 4 and 5 you genetically engineered cells
with DNA plasmids that contained two genes, one that
coded for antibiotic resistance, and another to express
a trait such as a chromoprotein (color pigment). The
proteins that resulted from the K12 E. coli cells tran-
scribing and translating the genes had very different
functions:
1.
Chromoprotein (color pigment): This proteins
primary function is to be colorful. Once the protein
is created, it bumps around the cell and gives the
cell color. The color pigment DNA was initially
derived from a sea creature which benets in some
way from being colorful.
Antibiotic resistance: This proteins function is to
catalyze a chemical reaction. The protein created
for chloramphenicol antibiotic resistance is called
chloramphenicol acetyltransferase. The enzyme is
able to bind to the chloramphenicol molecule and
cause a chemical reaction that inactivates chloram-
phenicol and prevents it from being able to harm
the cell.
This chapter focuses on the latter, protein enzymes
that catalyze chemical reactions and how they are
used by scientists and genetic engineers to create
end-products. Steps 1 and 2 of the
Three Steps to
Microfacturing
are used to create proteins from a DNA
sequence (Figure 6-1), and, in many instances, the
protein itself is the end-product. However, in many
other instances, the protein is not the end-product, it
is instead used to catalyze a chemical reaction involv-
ing other molecules which lead to the desired end
product(s). This is what we consider Step 3 of the T
hree
Steps to Microfacturing
: the processing of molecules
using protein enzymes.
To explore the third step of microfacturing, you will
see rst hand in the hands-on exercises how enzymes
can be used to convert one molecule into another.
Using cell extract, you are going to make pure oxygen
using a naturally occurring protein enzyme called
catalase. You are going to complete a set of new genetic
engineering experiments whereby you engineer K12
E. coli cells to express protein enzymes that will help
you generate smells and a new set of colors in a test
tube!
In the Fundamentals section, we are going to dig deeper
into atoms, bonding, how enzymes function and the
underlying chemistry of enzyme processing. We will
then relate this topic back to cell metabolism and
how it relates to “Life. You will then fully realize that
the trillions of cells that make up you (and E. coli) are
packed full of enzymes, all of which are encoded by
genes stored in your cells’ genome. These enzymes
drive your metabolism, the chemical reactions that
keep you going.
A B
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148 Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Protein Product
like a colorful protein
Transcription
• initiation factors
• ribosome & rRNA
• amino acids/tRNA
• protein
• substrate (A)
• product (B)
Translation
Enzymatic processing
A B
DNA
is made of a string of deoxyribonucleotides
RNA is made of a string of ribonucleotides
Protein is made of a string of amino acids
subtrate
(A) &
product
(B) are
molecules
• sigma factors
• RNA polymerase
• ribonucleotides
STEP 1
STEP 2
STEP 3
Figure 6-1. The Three Steps of Microfacturing
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149Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Getting Started
Equipment and Materials
The Amino Labs’ Smell-it Kit
TM
and Blue-it Kit
TM
are contained in the Zero to Genetic Engineering Hero Kit
Pack Ch. 5-7 and include all the required pre-measured ingredients. These kits can also be ordered separately
at https://amino.bio/products
Shopping List
Exercise 1:
Wetware kit: Amino Labs Smell-it Kit
TM
(https://amino.bio)
Exercise 2:
Wetware kit: Amino Labs Blue-it Kit
TM
(https://amino.bio)
Microcentrifuge
Small scale
Exercise 1 & 2:
Minilab (DNA Playground)
Microwave
Post-practice exercise:
Standard hydrogen peroxide (available at pharmacy)
Cell extract from the Blue-it Kit
Instructions Overview (for both Exercises)
Day 1-4 - “Bag 1”
1. Complete the genetic engineering exercises in “Bag 1”
Day 4-6/7 - “Bag 2”
2.
Complete the culturing exercises using “Bag 2” to obtain cultured cells or cellular extract containing
the engineered protein enzyme.
3. Add substrate to the experiment to complete Enzymatic Processing.
Chapter Timeline Overview
Timeline to complete each hands-on exercise is:
Day 1: ~60 minutes followed by 12-24 hours incubation,
Day 2: ~60 minutes followed by 12-24 hours recovery
Day 3: ~30 minutes followed by 24-48 hours incubation
Day 4: ~45 minutes followed by 24-48 hours incubation
Day 5: ~45 minutes followed by 24-72 hours incubation
Day 6: ~45 minutes
Day 7: ~45 minutes (Blue-it kit only)
Timeline to read Fundamentals is typically 3 hours.
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150 Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Learning Hands-On: Process one molecule into another
using enzymes
You’ve learned the basics of how to grow, engineer, culture, and lyse cells to obtain a cell extract that contains
your microfactured proteins. We are now going to put those skills to work! In these hands-on exercises, you are
either going to culture engineered cells and process molecules within the petri dish, or you are going to use
your cell extract to convert one type of molecule, called a substrate, into a different molecule, called a product
(Figure 6-1).
Chapter 6 includes two different exercises that demonstrate different ways to do enzymatic processing. Exercise
1 involves engineering bacteria to create an enzyme called Atf1 that converts a substrate into another with an
overripe banana smell. This will be done within cells in the petri dish. Exercise 2 includes engineering cells to
create an enzyme called beta-galactosidase that converts a colorless substrate into a blue product. This will be
done in using cell extract.
Exercise 1: Enzymatic processing to generate smells
Step 1. Download the instruction manual for the Smell-it Kit
Familiarize yourself with the Smell-it Kit instructions at https://amino.bio/instructions. The manufacturer’s
instructions will have the most up to date procedures for this exercise.
Warning: This experiment includes odors! If you are sensitive to odors or perfumes (give you headaches, make you
nauseous, etc.), the odors in this kit could trigger your sensitivity.
Step 2. Complete the engineering part of the Smell-it Kit
Complete the Smell-it Kit “Bag 1” genetic engineering portion of this exercise just as you have in Chapter 4.
Whereas in prior genetic engineering experiments your primary objective was to engineer cells to produce
color pigments, in this exercise you will engineer the cells to produce a protein enzyme. Upon successfully
engineering the cells, they will appear visually similar to the blank cells that you streak on the non-selective LB
agar plates. This is because the enzyme does not have a color. Do not be fooled! If they grow on selective plates,
the plasmid is being expressed.
Step 3. Culture cells with the substrate
Within the Smell-it Kit “Bag 2” you will nd the culturing materials and tubes of substrate. The substrate is
called isoamyl alcohol and has a musky smell. Open up the tube and gently waft your hand over the top toward
your nose to try to smell the liquid. (Look at the Smelling reagents Pro-tip coming up if you are not familiar with
wafting). Put the lid back on until you complete the next steps.
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151Zero to Genetic Engineering Hero - Chapter 6 - Processing Enzymes
Step 4. Labeling and creating your LB agar plates for culturing
A. You will use four selective LB agar plates for the culturing part of the Smell-it Kit experiment. Label them:
E. coli + Atf1 + IA; Chlor (E): This is your experimental sample. If everything is done correctly, you should
smell overripe banana. The organism is E. coli expressing Atf1. The plate will contain the substrate isoamyl
alcohol (IA). The antibiotic is chloramphenicol.
E. coli + IA; Chlor (- control): This is a negative control. The organism is negative control E. coli cells and
does not express Atf1. The plate contains the IA substrate. The antibiotic is chloramphenicol. These cells
will grow on selective LB agar plates, but will not express the Atf1 protein and therefore won’t process the
substrate. Because there is substrate, the plate should smell similar to the original substrate in the tube.
E. coli + Atf1 + IA; Chlor (+ control): This positive control includes you streaking a plate with positive (+) cells
included in the kit that express Atf1. You will add IA to the plate. Similar to the other plates chloramphenicol
is the antibiotic for selection.
E. coli + Atf1; Chlor (- control): This is a second negative control. In this instance, you will streak your plate
with some Atf1 expressing engineered E. coli (+ cells). However, you do not add IA substrate. This control will
verify that the E. coli themselves do not produce the overripe banana smell.
Figure 6-2. Label your petri dishes for your experimental sample and three controls
Figure 6-3. Transfer IA into petri dishes, and slide the paper disk over
top of the IA to trap it underneath.
Figure 6-4.
Pour molten LB agar into the petri dishes.
Use a yellow loop to hold the paper disks so they won’t
oat away.
Smelling reagents Pro-tip
When smelling materials in any lab or engineering environment, you should never place your nose directly
over the top of the container opening and inhale. This is very dangerous because the material you want to
smell could be toxic or overwhelming. Wafting is a safe technique to use when smelling reagents.
Visit: https://amino.bio/wafting
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