101Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
Fundamentals: How a cell reads a DNA plasmid
The basic operating environment
of a cell: The Four B’s
(Bump, Bind, Burst, Bump)
In a large red brick factory, raw materials are turned
into products using machines that rely on workers
who pull levers and push buttons, or computers that
complete automated protocols through deliberate
and intentional actions. Does a cell “microfactory”
work like this? How does a cell know what to do?
Cells operate very differently than actual factories.
There are no deliberate or intentional acts by
atoms or molecules. Atoms and molecules do not
think or plan out what to do! Rather, three general
factors play a role in the ‘decision making’ or ‘logic’
of a cell:
• The number of molecules: Back in Chapter 1, we
learned that what’s inside a cell is a bit like a ball
pit - it’s packed full of molecules. These molecules
range from small ones like water to large ones
like a cell’s genome and protein machinery. Even
though this microfactory is very small, it will have
thousands or millions of copies of these cellular
machines to complete tasks. The quantity of any
particular atom or molecule determines how many
are available to participate in chemical reactions
or other processes. The more molecules or protein
machines, the more reactions happen.
•
The rate at which molecules bump into other
molecules: In general, atoms and molecules freely
move about the cell bumping into other atoms or
molecules at a very fast rate. In the world of the
very small, activities happen surprisingly fast.
For example, around you right now are trillions of
gas molecules – oxygen, hydrogen, nitrogen and
carbon dioxide – that ll the room. Do you know
how fast they are moving? Hydrogen moves at
more than 6,000 kilometers per hour - 60 times
faster than a car on a highway! These molecules
are so small that we don’t feel or notice them.
Further, the atoms that make up molecules can
vibrate at up to 100,000,000,000,000 times per
second (10
14
Hz). The fast movement and vibra-
tions mean that atoms and molecules can bump
into other atoms or molecules very often. Within
cells, molecules also move fast. Water molecules
have an average velocity of 2,000 kilometers/hour.
In other words, atoms and molecules can move and
vibrate at high speed over small distances.
Cells are packed full of different molecules which
vibrate and bounce around into one another very
rapidly. This allows for trillions or quadrillions of
interactions to happen in a single cell at any given
moment. A single molecule can bounce around
and interact with thousands or millions of other
molecules every second and, when chemical bond-
ing is strong enough between two molecules, a
chemical reaction may occur. Chemical bonding is
when atoms or molecules stick together.
•
The strength of chemical bonding between
molecules: Cell ‘logic’ and decision making is
based primarily upon chemical bonding, which is
the ability of some molecules to bind specically
or not with other atoms or molecules. This is what
causes certain chemical reactions that result in
product-making “actions” to take place.
These factors contribute to the basic operating envi-
ronment of a cell. In short, an extremely large number
of “events” involving a large number of atoms and
molecules in combination with bonding leads to an
action or outcome.
When thinking of the operating environment of a cell,
remember the Four B’s of Basic Cell Operation:
•
Bump: Molecules move fast and bump into other
molecules in the cell.
• Bind: When a molecule bumps into another mole-
cule, it can result in two or more molecules being
bound together if the chemical interaction is
strong enough.
•
Burst (optional): In the case of protein enzymes
that catalyze chemical reactions, when two mole-
cules interact, a ‘burst’ or change in energy may
occur resulting in a chemical reaction.
• Bump: The molecules or products of the chemical
reaction separate and continue bumping around
the cell.
These are by no means scientic terms, and there are
other mechanisms by which certain cellular opera-
tions occur, but the Four B’s of Basic Cell Operation are a
great starting point to understanding and remember
how E. coli cells work. The cells do not have a brain
and do not think in the way we understand thinking.
Instead, they use these Four B’s.
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102 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
The Three Steps to Microfacturing
Now that you understand the basic rules under which
cells operate, let’s look at how DNA can be ‘recog-
nized,’ ‘read’ and ‘executed’ by the cell. During the
hands-on exercise in this chapter, you genetically
engineered your cells with a DNA plasmid. Immedi-
ately after you put the DNA into the cells, they began
what we call the Three Steps to Microfacturing. Micro-
facturing is like manufacturing, except it happens at
the micro-scale or smaller. This is what scientists are
referring to when they talk about genetic expression.
We’ve seen that the order of nucleotides in a DNA
sequence make up the ‘blueprints’ for the cell. The
Three Steps to Microfacturing, describes three distinct
and separate processes that cells use to recognize and
read DNA sequences, ultimately decoding it to make
cellular products that have a function
(Figure 4-18).
•
Step 1 - Transcription: DNA is a stable chemical
molecule with unique sequences. Some regions of
DNA have a specic sequence that can be recog-
nized by cellular machinery. As you just learned,
this recognition happens when a chemical bond
that is strong enough exists between the cellular
machinery and the shape and physical character-
istics of the DNA segment. In the rst step of the
Three Steps to Microfacturing, DNA becomes bound
to and is ‘read’ by cellular machinery called RNA
polymerase. RNA polymerase travels along the
DNA reading it and simultaneously transcrib-
ing a similar looking, but different nucleic acid
string called ribonucleic acid (RNA). Just like
how a language translator can listen to Spanish
and translate to English in real-time, RNA poly-
merase reads DNA and simultaneously creates the
appropriate RNA molecule. We are going to cover
transcription and RNA in depth in this chapter.
Stochastic model of chemical reactions Web Search Breakout
The idea that molecules randomly move around cells and complete the Four B’s is generally called a
“stochastic process”, where there is a certain probability that a particular activity or chemical reaction
will happen. While this model does not need to be understood to perform good genetic engineering, you
can look it up online using terms like “stochastic model” and “collision theory”.
Restriction Enzymes Going Deeper 4-7
Restriction enzymes use the Four B’s. Restriction enzymes were some of the rst genetic engineering tools
used by scientists in the 1970s. They are protein enzymes that act like tiny molecular ‘scissors’ which cut
DNA at specic sequences. Restriction enzymes are proteins that have two functions:
1.
First, they bind to a specic region of DNA. The restriction enzyme bumps and jostles around the
cell, interacting with many other proteins, molecules, and parts of the E. coli genome. When a part of
the protein, called the binding domain, binds strongly enough with a region of the DNA molecule, the
protein will bind like a lock and key due to the width and physical characteristics of the DNA sequence.
2. The second function causes a chemical reaction that can break the chemical bonds of the sugar-phos-
phate backbone of the DNA’s double helix. That means the restriction enzyme can catalyze a chemical
reaction that cuts the DNA’s backbones. After a restriction enzyme “nds” the appropriate DNA sequence
(binding strongly to a region), it cuts the DNA. Once the DNA has been cut the shape of the DNA where
the restriction enzyme had bound changes. This change means the restriction enzyme is no longer
strongly bound to that DNA. The DNA and enzyme separate, continuing to bump and bounce around
until it eventually bumps into another similar DNA sequence elsewhere in the cell.
In other words, the restriction enzyme doesn’t think, “I want to nd the DNA sequence GAATTC.” Instead, the
unique protein sequence and, therefore, the unique amino acid structures (Chapter 3) can specically bind
to the GAATTC DNA sequence. The restriction enzyme bumps around the cell interacting with millions or
trillions of other molecules until it interacts with a GAATTC sequence. It then completes its scissor function.
It can be argued that all molecules created in the cells have a specic function that the cell has evolved them
to have. Combined, these individual functions add up to a very complex machine that seems to have logic.
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103Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
•
Step 2 - Translation: As you will learn in the
coming sections, RNA is a less stable chemical
molecule than DNA, but both molecules share
many similar characteristics. In step two of the
Three Steps to Microfacturing, another kind of protein
machinery called a ribosome recognizes a region
of the RNA through chemical bonding, along with
help from a distinct type of RNA called transfer
RNA (tRNA).
The ribosome and tRNA read the RNA, simultane-
ously translating the RNA sequence into strings
of amino acids called proteins. As you learned in
Chapter 3, proteins make up the vast majority of
cellular machinery. For example, in this chapter’s
hands-on activity, the color pigment produced by
your cells was likely a protein. The antibiotic resis-
tance was also due to a protein. Translation, tRNA
and protein creations will be covered in Chapter 5.
•
Step 3 - Enzymatic Processing: The third step
of the Three steps to Microfacturing is Enzymatic
Processing. This includes how protein enzymes
cause the chemical reactions that make life
happen. We will cover it in Chapter 6.
Now, let’s have a deeper look at Step 1, transcription.
To do so, we will rst learn about a very important
nucleic acid, ribonucleic acid, or RNA.
Deoxyribonucleic acid (DNA) vs.
Ribonucleic acid (RNA)
During transcription, cell machinery reads DNA and
simultaneously creates a “sister molecule” called
RNA. What is RNA?
An RNA strand looks a lot like a single strand of DNA.
The main difference is that the nucleotides making up
RNA are very slightly different from DNA nucleotides.
Look at Figure 4-19. Can you spot the difference?
If you closely compare the ribose sugar ring of the
nucleotides, you’ll notice that there is a slight differ-
ence at the “bottom” of the ring. The ribonucleotide
(RNA) has two “OH” groups - one on the C3 carbon
atom, and one on the C2 carbon atom. The DNA’s
deoxyribonucleotide has only a single ‘OH’ group,
with the C2 carbon instead having a hydrogen ‘H’.
Deoxy, the removal of the ‘oxy’ or oxygen atom at the
C2 position, is why DNA is called deoxyribonucleic
acid. RNA, on the other hand, has two OH groups on
the ribose sugar and is referred to as ribonucleic acid.
Beyond this slight difference in the ribose sugar ring,
the ribonucleotides of RNA connect together in the
same way as DNA (Figure 1-17). The OH on C3 of one
nucleotide ribose sugar connects to the phosphate on
C5 of another, and this repeats to create a sugar-phos
-
phate backbone. Notice that when two nucleotides are
connected, the “H” on the “OH” is removed during the
reaction (Figure 4-20).
A second difference between DNA and RNA is that
RNA does not form a double helix structure like
DNA. As you’ll see at the end of this chapter, RNA
Figure 4-18. The Three Steps to Microfacturing includes DNA
being transcribed into RNA, and RNA being translated into
proteins. Becoming a Genetic Engineering Hero means
knowing how all of these steps work so that you can control
what the cell makes, when the cell makes it, and how much
it makes!
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
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104 Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
nucleotides can complement and bind to one another
and form structures, but this happens at a much
lower frequency than DNA. This is why in Figure 4-18,
the RNA is a lone single strand, while DNA is illus-
trated as a double helix.
This slight change in the ribose sugar and the fact that
it doesn’t broadly form a double helix with comple-
mentary RNA has profound effects on RNA’s stability
and function. DNA is very stable. So much so that it
can remain intact for millions of years. RNA, on the
other hand, is not very stable. Once the cell creates
RNA, it stays intact for only a short time before falling
apart in minutes or hours.
A four-nucleotide string of RNA can be found in
Figure 4-20. You’ll see it has a very similar structure
to a single strand of DNA. One difference is that the
RNA nucleotide has the extra “OH” hydroxyl group
in the ribose sugar on carbon C2. Another difference
is that RNA does not have thymine. Instead, it has
“uracil” (U) nitrogenous base in the uridine ribonu-
cleotide.
Now that we know more about the structure of RNA,
we can learn about transcription, the process that the
cell uses to read DNA and transcribe it into RNA. To
do this, we are going to look at what a gene is and ask
these questions:
• What is the cell machine that catalyzes the chemi-
cal reaction during transcription?
• How does it know where to start transcribing?
• How does it know what to transcribe?
• How does it know when to stop transcribing?
Figure 4-19. Comparing the nucleotide of DNA (left) and RNA (right), you’ll notice only a very minor difference in the structure.
Ribonucleotide
Deoxyribonucleotide
O
C
OH
C
C
C
Phosphate
Nitrogenous
base
C
H
can bind to
phosphate
O
C
OH
C
C
C
Phosphate
Nitrogenous
base
C
OH
can bind to
phosphate
1
23
4
5
1
23
4
5
Figure 4-20. Four nucleotide string of RNA.
O
O
C
CH
2
O
O
C
P
N
O
C
C
NN
C
C C
C
H
H
-O
O
C
CH
2
O
O
-O
O
C
P
O
C
C
N
C
C
N
H
N
H
H
C
C
N
N
C
H
O
C
CH
2
O
O
C
P
N
C
C
O
NC
C
C C
N
H
H
N
N
C
O
-O
O
C
CH
2
O
O
C
P
O
C
C
O
NC
C
C C
N
H
-O
O
H
A
Adenosine
C
Cytidine
U
Uridine
G
Guanosine
5’ Phosphate end
3’ OH end
O
H
O
H
O
H
O
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105Zero to Genetic Engineering Hero - Chapter 4 - Genetic Engineering Your E. coli Cells
RNA polymerase: The cell
machine that transcribes
The cellular machine responsible for transcription is
a protein enzyme called RNA polymerase.
RNA: because it catalyzes the creation of RNA
Polymerase: because it joins RNA nucleotides (ribo-
nucleotides) together into a string. Polymer is a
general chemistry term to describe a chemical string
of one or more reoccurring building blocks.
Just as RNA polymerase is involved in expressing the
genes that were produced by your engineered E. coli,
RNA polymerase itself is expressed from a gene in
the genome of the cell. That means RNA polymerase
is involved in creating itself!
As you’ll see later in this chapter, RNA polymerase is
able to bind to a DNA strand (Figure 4-21). It can then
ride along the DNA like a train on tracks, and simulta-
neously create (transcribe) an RNA molecule.
Compared to the width of the DNA strand, RNA poly-
merase is much wider and is able to surround the
strand of DNA. In Figure 4-22, the RNA polymerase
(pink) surrounds a piece of DNA (orange/blue). Unlike
the artist’s depiction in Figure 4-21, Figure 4-22 is a
mathematical model based on real data of RNA poly-
merase bound to DNA. So while you don’t see a full
DNA strand passing through the RNA polymerase,
this illustration provides a very real perspective on
the size of RNA polymerase compared to DNA.
In coming sections, we will go into much deeper detail
about how RNA polymerase knows where and when
to start and stop transcribing.
Chemical Nomenclature Pro-tip
How do you number the carbons in molecules such as deoxyribose? The simple rule for numbering a
molecule’s carbons is that the #1 carbon (C1) is the carbon with the most atomic weight attached to it. Have
another look at the periodic table at the end of the book, and you’ll see the atomic weight of each atom,
carbon for example, has an atomic weight of 12.011 g/mol.
Carbon 1 of deoxyribose has an oxygen (15.999 g/mol), a carbon (12.011 g/mol), a nitrogen (14.007 g/mol)
and an hydrogen ((1.008 g/mol) attached to it. This totals 43.025 g/mol. Calculate some of the other carbons
in the ribose ring to see what the atomic weights of their bound atoms add up to. You’ll nd that C1 has the
highest atomic weight attached to it.
Once you’ve identied the carbon attached to the highest atomic weight, you designate it carbon 1 (C1). The
next neighboring carbon is C2, and so on. Because C1 only has one neighboring carbon, the next carbon is
C2. If there were carbons on each side, you would continue to number toward the next carbon with a side
chain of the second highest molecular weight.
Figure 4-21.
Artist view of RNA polymerase protein (blue green)
bound to a DNA strand (orange) and transcribing RNA (red).
Source:
Protein Data Bank (PDB) Bruce Alberts, A.Johnson, J. Lewis, M. Raff, K. Roberts
and P. Walter (2002) “Molecular Biology of the Cell” Ch. 6, Garland, New York.
Figure 4-22. Crystal structure of RNA polymerase (pink)
bound to a DNA helix (orange/blue). When operating within
the cell, the DNA continues through RNA polymerase, and an
RNA strand protrudes out the surface. Source: Ibid
DNA
RNA polymerase
RNA
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