182 Zero to Genetic Engineering Hero - Chapter 7 - Manually turning on genes in situ
ON
OFF
on completing your sixth set of experiments!
37 °C with the light on and off by following the direction in the instructions manual.
For this system, the wavelength of light is critical. The CcaSR system needs 535 nm light to become activated.
If your DNA Playground has the built-in LED module, turn it on. If you are using the standalone Light-it LED
chamber, connect the battery by following the instruction manuals, then place it inside a 37 °C incubator. Note
that accurate temperature is important for this induction to work.
Light and bacteria Going Deeper 7-3
Light is an essential sensory cue for many organisms. You use your eyes to sense light that is reected off
of the environment around you. Insects and animals use similar methods. Plants use light to understand
growing seasons. Bacteria use light to understand the environment around them.
Phytochromes are proteins in plants and bacteria that are able to sense light radiation in many different
wavelengths. The light may be visible, such as in the blue, green, or red spectrum, but may also be in the
far red and infrared spectra (heat). In addition to sensing the light, phytochromes have the ability, through
many different mechanisms, to activate and repress gene expression.
In the case of this exercise, the phytochrome CcaSR is from a genus of water-dwelling cyanobacterium, called
Synechocystis that use it to regulate their circadian rhythm, and for phototaxis (movement based on light).
Figure 7-1. Use the built-in LED in your DNA Playground Large or the standalone Light-it LED chamber to shine light on your plates.
R-BG
S.
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183Zero to Genetic Engineering Hero - Chapter 7 - Manually turning on genes in situ
Fundamentals: Diving deeper into genetic ‘switches’
There are hundreds of known and countless
unknown genetic “switches” that enable cells to turn
gene expression on or off based on the environmen-
tal conditions outside of the cell, or the physiological
state inside of the cells. In the exercises within this
chapter, you explored three different classes of gene
regulation - chemical, temperature, and light. There
are many more gene regulation systems, including
many that are unknown to the authors!
In this section, we are going to explore more in-depth
how each of the gene regulation pathways used in the
hands-on exercises works, and relate their function
back to the original operation that occurs in the cells.
Turn on genes with chemicals
Chemicals can be broadly dened as any molecule.
Genes can be turned on or off by many different mole-
cules ranging from molecular oxygen gas, to sugars,
and even heavy metals like arsenic.
In Exercise 1, the genetic system you used was
inspired by a genetic regulatory network called the
lac operon. The lac operon was discovered nearly 100
years ago in E. coli bacteria and it unveiled for the rst
time the sophistication of genetic circuits in cells.
However, rather than look at how the entire lac operon
genetic system functions, we will focus on one part
which you used in the hands-on. The critical mole-
cules that are involved in the chemical induction
exercise include:
Gene of Interest:
1. The coding region of the gene contains the DNA
sequence for colored pigment. When transcrip-
tion and translation occur, colored proteins are
created. In Chapter 5 you learned that in transla-
tion, the coding region has a start codon at the 5’
end and a stop codon at the 3’ end.
2.
The non-coding region of the gene is where the
magic happens in this exercise. You are familiar
with promoters (Chapter 4) and the ribosomal
binding site (Chapter 5). They are important for
RNA polymerase and ribosome binding, which
initiates transcription and translation. In this
genetic circuit, there is also a short sequence
within the promoter called an operator. Similar
to how the promoter and RBS are able to bind to
proteins such as RNA polymerase and the ribo-
some, the operator is a short segment of DNA that
is able to bind to other proteins. These proteins can
either activate gene expression with an ‘activator’
(similar to a sigma factor) or repress gene expres-
sion with a ‘repressor’. The repressor represses
gene expression by preventing RNA polymerase
from binding to DNA or initiating transcription. In
the exercise you completed, there is a repressor
called the lac repressor which is able to bind to the
operator tightly and stay there (Figure 7-2).
Lac repressor: Lac repressor is a protein that is
also expressed in the plasmid that you engineered
into your K12 E. coli. Lac repressor is capable of
Figure 7-2. After transforming your cells, your gene of inter-
est is: 1) in the ‘off’ state because lac repressor is bound to the
lac operator; 2) the added IPTG inducer binds to lac repressor
which changes its shape so that it can no longer bind to the lac
operator and block RNA polymerase; 3) with the lac repressor
removed, the RNA polymerase initiates transcription.
Promoter
1.
2.
3.
RBS
Coding sequence - colour pigment
Promoter
Lac
Operator
Lac
Operator
Lac
Operator
Coding sequence - colour pigment
Promoter RBS
RBS
Coding sequence - colour pigment
RNAP
RNAP
RNAP
Lac Repressor
Lac Repressor
IPTG
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184 Zero to Genetic Engineering Hero - Chapter 7 - Manually turning on genes in situ
binding tightly to the lac operator. When bound, the
lac repressor acts like a roadblock, preventing RNA
polymerase from initiating transcription, meaning
that gene expression cannot occur. (Figure 7-2, 7-3).
Inducer: IPTG is a small chemical molecule that is
used as an inducer, meaning it can cause induction
(activation) of gene expression. IPTG is a molecule
synthesized in laboratories that is similar to a natural
molecule called lactose (Figure 7-4).
The natural inducer, lactose, is a sugar that acti-
vates gene expression by binding to lac repressor. In
the natural lac operon in cells, the protein enzyme
created upon activation of gene expression is called
beta-galactosidase. Beta-galactosidase is a protein
that ultimately cuts the lactose sugar in half to become
glucose and galactose (Figure 7-5).
In other words, the lactose molecule activates gene
expression to create an enzyme that destroys it! When
it destroys the lactose, the galactose and glucose
become a carbon energy source for the cell. The prac-
tical reason for this is that the cell does not want to
create enzymes when they don’t need them. Why
should the cells always create beta-galactosidase if
there is no lactose present? Only when lactose is pres-
ent will it create the enzymes to break the lactose into
a usable food source. Recall in the Blue-it Kit (Chapter
6), you engineered cells to create beta-galactosidase,
the same enzyme that originates from the lac operon!
Now that you know the natural function of the lac
repressor, lac operator, and lactose inducer, you may
realize that the lactose inducer isn’t really the ideal
inducer for genetic engineers. This is for two reasons:
1. Lactose can be consumed by bacteria as an energy
source. This means that when you add the inducer
to the system, the cells will slowly consume lactose
until none is left and induction stops.
2.
Because lactose is an energy source for cells, by
adding it, you are changing the cells’ metabolism.
This can create variation in how the cells operate
and the results of your genetic engineering exper-
iment might be more variable.
IPTG was created by scientists to overcome both of
these issues. IPTG is not consumed by the cell, and
therefore it cannot directly influence the energy
systems of the cell. Second, because IPTG cannot be
consumed by the cell, the number of IPTG molecules
that you add to the system remains constant. This
enables much better control of the induction of genes.
To summarize the activity that occurs when you
induced gene expression with IPTG:
1.
After you genetically engineer the E. coli with the
plasmid, the gene of interest is in the ‘off state’. This
is because lac repressor, which is constitutively
expressed (automatically turns on) from another
gene in the same plasmid, binds to the lac operator
of your gene of interest, blocking RNA polymerase
from initiating transcription.
2.
Upon adding IPTG inducer to the petri dish, the
IPTG crosses the cell membrane into the cell
cytoplasm and completes the Four B’s. When it
interacts with lac repressor, it is able to bind, and
this changes the shape of the lac repressor, causing
it to detach from the lac operator.
3.
With the lac repressor removed from the gene of
interest, the RNA polymerase can initiate tran-
scription.
Isopropyl-
thiogalactoside
(IPTG)
OH OH
HO
HOH
2
C
O
S
H
3
C
CH
3
Lactose
OH
OH
OH
CH
2
OH
O
O
OH
OH
HO
CH
2
OH
O
Figure 7-4. Lactose is the natural sugar inducer that binds
with lac repressor to remove it from blocking transcription.
IPTG is a chemical synthesized by scientists that is much
more stable than the natural inducer and can be used in place
of lactose
Figure 7-3. Lac repressor (green) is a protein with ‘two feet’
that can bind tightly to the lac operator which can be embed-
ded within the promoter of a gene. This action prevents the
RNA polymerase from proceeding with transcription. Source:
D. Goodsell, S. Dutta, C. Zardecki, M. Voigt, H. Berman, S. Burley. (2015)
The RCSB PDB “Molecule of the Month”: Inspiring a Molecular View of
Biology. PLoS Biol 13: e1002140.
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185Zero to Genetic Engineering Hero - Chapter 7 - Manually turning on genes in situ
Lactose
OH
OH
OH
CH
2
OH
O
O
OH
OH
HO
CH
2
OH
O
ß-Galactosidase
H
2
O
OH
OH
OH
CH
2
OH
O
OH
HO
OH
OH
HO
CH
2
OH
O
Galactose Glucose
+
Figure 7-5. In the natural lac operon, upon removing the lac repressor, the enzyme beta-galactosidase (beta-gal) is expressed. Be-
ta-gal has the function of cutting lactose between its sugar rings, resulting in galactose and glucose.
RNAP RNAP
σ70 Promoter
1.
RBS Coding sequence - colour pigment
σ70
σE Promoter
2.
RBS
Coding sequence - colour pigment
σE
Figure 7-6. Using the cells natural sigma factor expression to control a gene of interest. Left: Under favorable cell operations, sigma
70 recruits RNA polymerase to initiate transcription of your gene of interest; Right: Under stress such as heat, the cell creates sigma
E, which can be used to initiate transcription of your gene of interest.
Turn on genes with temperature
In Chapter 4, you learned that to start transcription
you need a promoter that sigma factors bind to, which
then “recruit” or bind to RNA polymerase to initiate
transcription. E. coli cells have many different sigma
factors that are expressed in the cell under many
different conditions and are always working in the
background to make sure that essential cellular stuff,
such as tRNAs and polymerases, are being created.
For example, under normal cell operation, where
the cells are grown in a favorable environment at 37
°C, with plenty of food, a sigma factor called sigma
70 (σ70) will be automatically expressed by genes in
the cell. σ70 is responsible for causing expression of
many “housekeeping” genes that enable overall cell
survival and growth.
In the plasmids you have been using for your genetic
engineering exercises, many of the constitutive
promoters that caused expression of the genes of
interest, such as color pigment genes, are recognized
and bound to by σ70.
If the cell encounters starvation or unfavorable
environmental conditions such as heat, other sigma
factors are expressed. These sigma factors are able
to cause expression of a different set of genes that
are typically off, to aid with helping cells to survive.
Incubating E. coli bacteria at temperatures of 42 °C
to 50 °C is very stressful to the cells and causes the
expression of such sigma factors. σE is a sigma factor
that is related to E. coli temperature stress response.
By incubating the cells at 42°C and above, a signi-
cant change happens in the cells, and they begin to
use a sigma E (σE) to cause expression of genes. The
plasmid you used in this exercise uses σE promoter
so that when the temperature increases in the cells’
environment, σE can bump into and activate expres-
sion of your color gene. (Figure 7-6).
In the case of the exercise you completed, it is as
simple as using the cells natural response to danger
or stress to your advantage. Unfortunately, E. coli cells
do not enjoy high temperatures, nor can they survive
for extended periods of time, so using this strategy as
a long-term tool is not ideal. E. coli cells can survive
at up to 46 °C for prolonged periods of time, but
anything hotter will lead to their demise.
37 °C 37-42 °C
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186 Zero to Genetic Engineering Hero - Chapter 7 - Manually turning on genes in situ
Turn on genes with light
Light is one of the most important environmental cues
for cells and all living organisms. Being able to see
physical objects in your environment at a distance is
key to survival for many organisms. Many organisms
use light as an energy source. For example, plants and
bacteria photosynthesize. While these organisms are
harnessing light as an energy source, they can also
learn about and respond to their environment. They
can measure the amount of certain colors of light.
Responding to the environment, whether you are a
human or a bacteria, involves light photons hitting
molecules or macromolecules either on or inside the
cells. Those molecules may harness energy from the
light photons to change shape or to cause a chemi
-
cal reaction to happen. In the case of the hands-on
exercise where you induced gene expression using
light, there are several different cellular systems at
play. The plasmid that your E. coli were engineered
with includes several different players. The following
section is an in-depth discussion of the light induction
system.
CcaS: is a protein that is constitutively expressed in
the cell after engineering the cell with a plasmid. CcaS
has two parts, called domains, that have two different
functions. One is the light receptor domain, which
is able to absorb green light of ~532 nm. The second
domain is called a kinase (k-eye-naise). A kinase has
the function of adding phosphate (PO
4
) molecules
to other molecules in the cell, such as proteins. As
you have seen throughout this book, phosphate is
highly negatively charged. By adding a phosphate to
a protein, the negative charge will change the shape
of the protein through ionic bonding and hydrogen
bonding. When the shape of a protein changes, it can
be activated to complete a chemical reaction, or inac-
tivated to stop completing a chemical reaction.
When the light receptor domain absorbs light, the
kinase domain of the protein becomes active and is
able to phosphorylate (add a phosphate to) another
protein in the cell. The protein that becomes phos-
phorylated in this system is called CcaR.
CcaR: is a protein constitutively expressed in the cell
upon transforming the cell with the plasmid. CcaR has
two domains as well. One domain is able to specically
bind to CcaS so that it can become phosphorylated.
When CcaR becomes phosphorylated, its shape
changes so that the other domain can bind speci-
cally to a promoter called pCpcG2-172. CcaR can act
like a sigma factor and cause RNA polymerase to bind
and initiate transcription. In the plasmid you used,
the light activation of CcaR causes the expression of a
second sigma factor called CCG.
Sigma CCG: In the plasmid that was pre-engineered
into E.coli cells, the pCpcG2-172 promoter is placed in
front of a coding sequence for another transcription
factor that will activate the expression of your gene of
interest. In this case, the protein CCG is able to bind
the pCCG promoter, which causes transcription of
your gene of interest.
Core T7 polymerase: T7 polymerase is a very popular
polymerase that genetic engineers use to selectively
transcribe coding sequences. T7 polymerase is an RNA
polymerase that comes from a bacterial virus called
a phage. It can transcribe from a specic promoter
called a T7 promoter because it has a ‘built-in sigma
factor’ that will bind to the T7 promoter. This means
that no E. coli bacteria will naturally create T7 poly-
merase, and nor will the cells be able to transcribe
from a T7 promoter. In the case of the plasmid you
used in this exercise, the T7 polymerase has been “cut
in half” so that only the transcribing part of the T7
polymerase is constitutively created by the cell. The
genetically engineered sigma factor called sigmaCCG
is expressed due to CcaR binding to pCpcG2-172.
CcaR binding to pCpcG2-172 happens when the
right light is present. Sigma factor CCG binds to the
CCG promoter, pCCG, which is at the start of your
color-producing gene. The T7 Core polymerase binds
to sigma factor CCG and transcribes your the coding
sequence for your color pigment.
This is a pretty complex genetic pathway, but it will
illustrate the sophistication of genetic regulation in
cells and that there is a lot of potential for innovating
in genetic engineering (Figure 7-7)! To summarize the
light activation system:
1. As in other plasmids you’ve used, the antibiotic
selection gene is designed to automatically create
antibiotic resistance so you can select for your engi-
neered bacteria.
2. Rather than using the cell’s natural RNA poly-
merase, you are using a unique polymerase called
T7 polymerase that will be specic to the genes you
want to express in the plasmids in the pre-engineered
bacteria. Automatic expression is used so that the
RNA polymerase is ‘ready and available’ for transcrip-
tion of the color-producing gene in the plasmid.
3-4. Light sensor proteins CCaS and CCaR are also
automatically expressed so that they can be ready for
when the right light is present.
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