177Zero to Genetic Engineering Hero - Chapter 7 - Manually turning on genes in situ
Chapter 7
Manually turning on genes in situ
Congratulations! You are well on track to becoming
a Genetic Engineering Hero! Your ability to complete
fundamental techniques like making LB agar
plates, transforming E. coli, incubating cells and
even extracting enzymes and proteins are reaching
post-graduate university levels. In fact, many grad-
uate students have never made LB agar plates! In this
chapter, we’re going to build on the information and
skills you’ve learned and practiced throughout the
book to begin controlling E. coli cells after they have
been genetically engineered.
Up until now, you have been engineering cells with
plasmids that automatically operate. The cells
automatically copied the plasmids, and the genes
automatically turned on. These features were built-in
to ensure the hands-on exercises and Fundamentals
were within grasp.
This approach enabled you to complete the Funda-
mentals sections in Chapters 4 and 5, so you could
understand how transcription and translation start,
run, and stop. You may want to jump back to review
those topics as they are essential to understand once
you start manually operating genes and become a
genetic engineer.
In this Chapter, you are now going to complete three
different exercises teaching you how genes can be
turned on manually using various mediums such as
chemicals, temperature, and light. The technical term
for turning on genes manually is ‘inducing expres-
sion’. Inducing expression is an instrumental skill to
add to your genetic engineering toolkit.
In the Fundamentals section, we’re going to look
deeper into how gene regulation works. While you
will not be creating your own DNA plasmids, this
chapter will provide you with some deep insights into
what you should be thinking about when you do start
designing and building your own plasmids and genes.
We will relate what you learned in Chapters 4-6 to how
the “automatic gene regulation” works and then look
at how manual gene regulation works.
ON
OFF
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178 Zero to Genetic Engineering Hero - Chapter 7 - Manually turning on genes in situ
Getting Started
Equipment and Materials
The Amino Labs Light-it Kit
TM
, Heat-it Kit
TM
, and Induce-it Kit
TM
are part of the Zero to Genetic Engineering
Hero Kit Pack Ch. 5 - 7. These kits can also be ordered separately at https://amino.bio/products
Shopping List Choose one of, a combination of, or all exercises:
Exercise 1:
Wetware kit: Amino Labs Induce-it Kit
TM
(https://amino.bio)
Exercise 2:
Wetware kit: Amino Labs Heat-it Kit
TM
(https://amino.bio)
Exercise 3:
Wetware kit: Amino Labs Light-it Kit
TM
(https://amino.bio)
The Light-it-enabled DNA Playground Large or the standalone Light-it LED add-on
Exercise 1, 2 & 3:
Minilab (DNA Playground)
Microwave
Instructions Overview
Day 1-4
1. Complete the Engineer-it Kit exercise(s) as required to obtain engineered cells. (Note: The Light-it
Kit does not require engineering of cells).
Day 4-5
2. Culture your cells and manually turn on gene expression:
A. Chemical: Dissolve the inducer chemical using the dissolving buffer provided in the kit. Mix it
well and add the dissolved inducer to a region of the plate. The inducer will dissolve into the
LB agar. The area where you added the liquid drop will be the most induced and neighboring
regions will also be induced, just not as much. You will see a “gradient” of induction. You can
put drops of inducer in multiple locations.
B. Temperature: Place your engineered bacteria in the DNA Playground incubator initially at 30 °C
Then shift the temperature to 42 °C causing the cells to maximally express your trait.
C. Light: The color of light needed to induce expression is quite specic. Turn on the desired color
using the sliders on the DNA Playground Large or by using the standalone Light-it LED add-on.
Chapter Timeline Overview
The timeline to complete exercise 1 & 2 is:
Day 1: ~60 minutes with 12-24 hours incubation,
Day 2: ~60 minutes with 12-24 hours recovery
Day 3: ~30 minutes with 24-48 hours incubation
Day 4: ~45 minutes with 24-48 hours incubation
Day 5: ~45 minutes with 24-72 hours incubation
Day 6: ~45 minutes
The timeline to complete light induction with the
Light-it Kit is:
Day 1: ~60 minutes to make LB agar petri dish and
streak cells, with 24 hours incubation
Day 2: ~30 minutes to paint your living paintings with
24-48 hours incubation
Day 3: ~15 minutes to view results.
Day 3+: Repeat day 2/3 activities on any remaining
petri dishes
Timeline to read Fundamentals is typically 3 hours.
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179Zero to Genetic Engineering Hero - Chapter 7 - Manually turning on genes in situ
Learning Hands-On: Manually turning on genes in situ
In situ means “in position of”, and the hands-on exercises in this chapter involve rst engineering E. coli bacte-
ria with a DNA plasmid, and subsequently turning the desired genes on using an environmental cue while the
system is in the bacteria. Because the cells will not be programmed to turn on the desired gene(s) right away,
you’ll notice that the engineered cells look like normal E. coli cells - they are white in color. Only once you enact
the environmental cue will the cells begin the
Three Steps to Microfacturing
to create your desired end product.
In this chapter, you will use the skills you learned to genetically engineer cells. You can also complete some
of the other exercises you’ve learned along the way, like lysis and extraction; you just need an Extract-it Kit!
You would simply complete that exercise at the appropriate time, after you’ve induced and caused the cells to
express the end product. Would the end product be any different than in cells that auto-induce expression? That
sounds like a good experiment, you’ll have to try it out to see. This is what genetic engineers and scientists do!
Exercise 1: Inducing a gene using a chemical
Step 1. Complete the Induce-it Kit engineering exercise.
While in previous experiments your engineered cells would automatically express the desired trait, usually
in the “late stationary phase” of growth (see Going Deeper 5-1), the plasmid you are now using is in the mostly
‘off position’ until you turn it on. We say mostly off since all gene control has some leakiness, which means that
low/very low expression can happen even when the gene is “off”. Upon successfully engineering your cells,
they will be visually similar to the blank cells you streaked on the N.S. LB agar plates. Engineer the cells using
“Bag 1” of the Induce-it Kit.
Step 2. Culture your cells
Within the Induce-it Kit, there are two bags. “Bag 1”, which you used in Step 1, included all of the ingredients
needed for the initial engineering of bacteria with the chemically inducible plasmid. “Bag 2” contains the
materials and ingredients to culture your cells, as well as induce expression of a gene using an inducer. Using
the contents of “Bag 2”, culture your cells on selective media as you usually would. You may use the streaking
method you used with blank cells, or you can use the double streak method. Incubate your cells for 12-24 hours
at 37 °C.
Step 3. Add your inducer
A. Within the Induce-it Kit “Bag 2” you will nd a tube of inducer. This inducer is in powder form. Because
your kit was likely bumped around while in transit to you, the powder may have spread all throughout the tube.
To ensure you dissolve all of the powder, place the tube in your microcentrifuge and balance it. Spin the tubes
at maximum speed for 10 seconds. This will pull all of the powder to the bottom of the tube.
B. The Inducer is in powder form so you will need to dissolve it in liquid to add it to your petri dish with the
engineered cells. Using the pipet included in the kit, pipet all of the ‘dissolving buffer’ into the tube of inducer
powder. Pipet up (suck in) and down (squeeze out) 10 times to mix.
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180 Zero to Genetic Engineering Hero - Chapter 7 - Manually turning on genes in situ
C. Using a permanent marker, make one or more marks, like an X, on the bottom of the plate in the area(s)
where you want to put the inducer. This is where you will drop the inducer onto the plate.
D. Drop-by-drop, pipet all of the dissolved inducer onto your desired locations. The inducer will be at it’s high-
est concentration at these points. However, it will dissolve outwards in a circular pattern through the LB agar.
The higher the inducer concentration, the more the gene will be induced.
Inducer Going Deeper 7-1
The inducer in this kit is a small molecule called isopropyl β-D-1-thiogalactopyranoside, or IPTG for short.
IPTG mimics a sugar called lactose, the primary sugar in milk, which is used naturally by some cells (like
the E. coli naturally in your intestines). When lactose is present, it will induce lactose metabolizing genes so
that lactose can be used as an energy source.
The gene gets turned on by IPTG or lactose because IPTG binds to a protein called a repressor, which, in the
absence of IPTG, binds to the promoter of the gene. Because the repressor binds to the promoter region,
RNA polymerase cannot initiate transcription. When IPTG is added to the cells, it binds to the repressor
protein and causes a slight change in shape of the repressor which lowers its ability to bind to the promoter
region. The repressor ‘falls off’, allowing the RNA polymerase to initiate transcription (Figure 7-2).
Inducing cultures Pro-tip
In this exercise, you added the inducer after growing your engineered cells for 12-24 hours. Further, you
induced only on part of the plate. This was for demonstration purposes, allowing you to see some cells
exhibiting gene expression while others did not express the gene.
In many genetic engineering exercises, you would grow your engineered cells in liquid culture, and after
~12-16 hours of growth, add the inducer to the entire culture.
Exercise 2: Inducing a gene using temperature
Step 1. Complete the Heat-it Kit engineering
Similar to Exercise 1, the Heat-it Kit plasmid you engineered remains mostly ‘off position’ until you decide to
turn it on. The Heat-it Kit does not have as ‘tight’ switching as the Induce-it Kit. However, if you culture your
cells at ~30 ˚C, the gene will stay mostly off. Complete the genetic engineering portion of your Heat-it Kit as
you did in Chapter 4 using “Bag 1”. Continue on with “Bag 2” using the culturing technique you learned in
Chapter 5. After streaking the engineered cells on your plates, incubate them at 30 ˚C for 24 hours. After 24
hours you should see lots of growth. However, the colonies should be mostly white in color. You can also opt to
paint a picture with your engineered bacteria rather than streaking it!
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181Zero to Genetic Engineering Hero - Chapter 7 - Manually turning on genes in situ
Step 2. Increase the temperature
In the case of temperature-based gene induction, there are no external factors that need to be prepared, other
increasing the temperature of the environment. After your cells have grown and colonies are visible, you merely
increase your DNA Playground incubator to 42 °C.
Increasing the temperature Going Deeper 7-2
Increasing the temperature ‘stresses’ the cells. This causes a change in gene regulation throughout the
cell, including the production of some different types of sigma factors. Recall back to Chapter 4 when you
learned how sigma factors bind to the promoter of a gene and then to RNA polymerase. RNA polymerase
can then begin transcribing the DNA into RNA. Some types of sigma factors are not created by the cell when
the cell is in optimal growth conditions.
Optimal growth conditions include optimal temperature (37 °C), acidity/pH (~neutral), and other factors.
By increasing the temperature to 42 °C, the cell ‘realizes’ that growth conditions are no longer optimal and
begins creating sigma factors that will express genes to help the cell survive in the stressed environment.
For example, the promoter in the non-coding promoter region of the gene you are inducing is designed to
bind a sigma factor called sigma E. When the cell enters ‘stress mode’ it begins producing sigma E which
will bind to the promoter in your gene of interest facilitating transcription. In other words, this turns on
or induces expression of your gene. You are now ‘hacking’ the survival mechanism of the cell to produce
your proteins.
In some instances, if your cells grow into large colonies, or start dehydrating, this can activate the stress
response and cause induction of your gene.
Exercise 3: Inducing a gene using light.
The Light-it Kit does not require any engineering, and is similar to the Canvas Kit exercise you did in Chapter 3.
Complete the exercise as you did in the past by streaking a painting palette and using it to streak/paint pictures.
Step 1. Streak cells
Just like in Exercise 1 and 2, the cells you are now using are in the ‘off position’ until you decide to turn them
on. The cells will appear similar to the blank cells that you streak on the non-selective LB agar plates until you
choose to activate the genes with light.
Culture your cells on one petri dish to make your painting palette. You will incubate them under a specic light
to turn the color-producing gene on in the next step!
Step 2. Turn on the light!
In the case of light-based gene induction, there are no external factors that need to be prepared, other than an
environment with the appropriate wavelength of light. For the creation of your painting palette, you will streak
the Light-it cells and incubate them under the Light-it Kit LED (light emitting diode). After your cells have grown
and colonies are visible, you will use the painting palette to paint on your other petri dishes, and incubate at
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