Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 71
interference. The fence posts are anchored into the
soil using concrete (Figure 3-17).
In the microfactory, we also see a “fence line” at the
outer edge of our K12 E. coli cell. E. coli cells have an
outer perimeter called the “capsule-layer” or “slime
layer.” It has a similar function to a fence: to separate
the body of the cell from the outside environment.
The capsule layer is indeed slimy and protects the cell
by preventing chemicals (like antibiotics) and other
organisms from entering the cell or disrupting the cell
membrane. The capsule layer also determines how
well a cell can keep water inside.
Similar to how a factorys fence line is anchored to
the ground, the capsule layer of the E. coli is made of
strings of sugars that are anchored to the underlying
cell membrane by lipid macromolecules (Figure 3-17).
The hybrid molecules that make up the capsule layer
are made of both a sugar and a lipid. This molecule is
called lipopolysaccharide (LPS). Look back at gure
3-16 to see how the LPS sugar is anchored in the outer
membrane. We will talk more about lipids when we
visit the cell membrane. For now, we will investigate
what brings the “slimy” to the slime layer - sugars.
Sugars are part of a class of macromolecules called
carbohydrates. Carbo meaning they have lots of carbons
(C) and hydrates meaning they have lots of hydroxyls
(OH). These molecules are important. They make up E.
colis cell structure, and they are the ‘fuel’ that drive E.
coli cell metabolism. Carbohydrates are typically made
up of CHO, with carbon being the key atom that links to
other carbon, hydrogen and oxygen atoms.
Sugars can be made from a variety of carbons, hydro-
gens, and oxygens. One of the most commonly known
sugars, glucose, has six carbons that make up its
“backbone” (Figure 3-18 (left)) and, when dissolved
in water, a glucose molecule folds up to form a ring
structure (Figure 3-18 (right)). In Figure 3-18 (center),
you will find a glucose molecule that does not have
its carbons and hydrogens shown. See the Reading
Molecule Drawings Pro-tip below to learn more.
The hybrid molecule lipopolysaccharides (LPS) gets
its name from ‘lipo, meaning lipid (fat), ‘poly, mean-
ing many, and ‘saccharidemeaning sugar. Each LPS
is a string of sugar rings permanently connected to a
lipid anchor (Figure 3-16). In the cell’s capsule layer, it
is the strings of sugar rings that slip and slide on one
another, making the surface of the bacteria ‘slimy’.
Figure 3-18. Glucose is a simple sugar with six carbons that
are also connected to hydroxyl (OH) groups. On the left is a
linear depiction of glucose with all atoms visible. When glu-
cose is in liquid, like within a cell, it circularizes into the ring
structure on the right.
H
H OH
O
C
1
C
2
HO
H
C
3
H OH
C
4
H OH
C
5
OHC
6
H
2
OH
OH
OH
CH
2
OH
OH
O
H
OH
O
HO
OH
OH
OHCH
2
Reading Molecule Drawings Pro-tip
As a way to make drawing chemical molecules easier, scientists decided that, since carbon is a highly
used element in biochemistry, it could be left out of chemical drawings. When you see a molecule drawing
like Figure 3-18 (right), you must know that any vertices without any letters in the ring are carbons. For
example, the glucose in Figure 3-18 (left) has all of the elements drawn and labeled, and some, such as the
C1-C6 down the backbone don’t have to be labeled, as shown in Figure 3-18 (center). This may seem a little
complicated, but as you learn and practice chemistry, it will become easier.
Scientists took this further. Since they know the rules of what can bind to carbons (each carbon binds to
four other atoms in most circumstances, or requires four bonds), hydrogens are not annotated either. If you
look at Figure 3-18 (center and right), many hydrogens (H) are also missing. While they are actually present
in the molecule, they have been deliberately removed from the drawing for “simplication”. Every carbon
vertex” with an OH connected also has an H connected in the opposite direction (purposefully not shown).
Book _genetic engineering hero-AUG2021.indb 71Book _genetic engineering hero-AUG2021.indb 71 8/18/21 12:03 PM8/18/21 12:03 PM
Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 72
You may recall the reasons why lab strains of K12 E. coli
are safer to use: i) they cannot effectively share genetic
information and; ii) they do not have a phage infection.
Here is another reason: K12 E. coli are missing some
sugars in their LPS called the ‘O antigen. The O anti-
gen is usually part of the sugar ring string on the LPS,
serving as a rst line of defense for the cell against the
environment. Not having the O antigen means that
the rst defense layer of the microfactories is severely
disrupted and makes the E. coli highly susceptible to
dying from antibiotics, chemicals, acids, surfactants,
and dehydration. It is as though the factory fence is
only 1 foot tall with very large mesh holes. It offers
some protection but not great protection. For this
reason, K12 E. coli are not good at surviving outside the
petri dish and are great to use in experiments.
The Outer Wall (B)
Have a look at your tour map (Figure 3-16). We have
passed through the fence/capsule layer, and now we’re
moving on to the next stop - the outer factory wall.
The outer factory wall has one primary function - to
be a strong barrier between the inside of the factory
and the outside environment. The factory wall should
protect the interior from rain, snow, hail, and even a
tornado. The walls of the factory are made of solid red
brick that is reinforced with iron.
The outer factory wall in our E. coli is the outer cell
membrane (Figure 3-19). The function of the cell
membrane is not only to protect the cell from the outside
environment, but it also acts as a container for all activ-
ity inside the cell. Without the cell membrane, all of the
cell insides would spill out. There would be no cell!
As a general rule, the membranes of E. coli cells are
made up of lipids (Figure 3-20). Lipids are essential
for keeping cell structure. Lipids are made up of CHO,
CHON, CHONS, or CHONP, and in general, have the
common characteristic of having a “tail group” and a
“head group” (Figure 3-20), just like the surfactants
you used for cell lysis in Chapter 1.
The “head group” segment of the membrane lipid is
hydrophilic (water-loving). This means that this is the
part of the lipid that interacts with the water environ-
ment inside or outside of the cell. The head group is
hydrophilic because it is charged, making it interact
with water, which is also charged. The structure and
composition of the “head group” can vary greatly. For
example, in Figure 3-22, a negatively charged phos-
phate molecule is present. In other lipids, the phos-
phate is replaced by a sulfate or other chemical group.
The “tail group” segment of the membrane lipid is
hydrophobic (water avoiding) (Figure 3-20). This
means that this part of the lipid prefers not to make
contact with water. It is hydrophobic because it is
uncharged and does not like interacting with charged
molecules or atoms. Because of this, it prefers to inter
-
act with the tail groups of other lipids (Figure 3-20 and
3-21). This difference in binding preferences leads to
a structure called a lipid bilayer, where the water-lov-
ing head groups face outwards into the watery envi-
ronment, and the water-avoiding tails face inwards
toward other water-avoiding tails to form a protec-
tive barrier (Figure 3-20 and 3-21). When millions of
lipids are produced by a cell, the layer creates a robust
barrier that separates the inside of the cell from the
outside environment (Figure 3-16).
During the rst point of the tour, we talked about LPS
the chain of sugars that make up the slimy part of the
capsule layer. However, those sugars are bound to a
lipid anchor called “Lipid A. Lipid A has a hydrophobic
tail that ts snuggly with the other “tail groups” inside
the outer lipid bilayer at the surface of the cell. This
hydrophobic tail is the anchor that holds the
entire LPS
molecule in the membrane.
Figure 3-19. Brick barrier compared to a lipid bilayer barrier.
Figure 3-20. In membranes, the head groups are charged
molecules that interact well with water and other charged
molecules. The tail groups are non-charged, and they do not
like to interact with charged groups but like interacting with
other non-charged groups. For this reason, many biological
membranes are made of lipids that form of a lipid bilayer.
Head Group
hydrophillic
(+/- charged)
hydrophobic
(non-charged)
Tail Group
Book _genetic engineering hero-AUG2021.indb 72Book _genetic engineering hero-AUG2021.indb 72 8/18/21 12:03 PM8/18/21 12:03 PM
Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 73
Figure 3-21. Rather than brick or concrete, the cell membrane is made of lipids and sugars. Rather than doors that open and close,
proteins are embedded in the membrane and act as tunnels” that help specic molecules to enter and exit. The blueprints of the cell
are not made of paper, but DNA. Small proteins made of amino acids are the cell machinery and scaffolding of the cells and readily
interact with DNA. Flagella are a whip-like microstructure made of protein that help cells to move around.
Genome
Blueprint of the E. coli cell
Proteins
Floats around the cell
to perform chemical reactions
Protein machinery can
interact with DNA to
read it, write it and more
Flagella
Help the bacteria move
Embedded Membrane Proteins
Help to localize chemical
reactions and transport
molecules across membranes
Intermembrane space
includes peptidoglycan
Capsule layer
sugar with a lipid anchor
Outer membrane
lipid bilayer
Inner and outer membranes
The main barriers that make a cell a cell
Inner membrane
lipid bilayer
Book _genetic engineering hero-AUG2021.indb 73Book _genetic engineering hero-AUG2021.indb 73 8/18/21 12:03 PM8/18/21 12:03 PM
Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 74
The Lobby (C)
As we move into the factory, we pass through doors
into the lobby (Figure 3-16). Cells also have doors, in
a way. Some tubular-shaped proteins cross through
the cell membranes and act as “portals”, serving as
the entry and exit points of the cell. This is where food
enters the bacteria, and waste exits the cell. We will be
learning in-depth about proteins very soon.
Once you enter the lobby (Figure 3-23) you have also
entered the building. However, you are not yet on the
factory oor. The lobby is a transitional location and
acts as an extra checkpoint. In this lobby, there might
be a security guard keeping an eye open for any trou-
ble. The temperature, humidity, and air quality in the
lobby is controlled but may be different than that of
the factory floor. Nevertheless, it is much closer to
those conditions than the outside environment.
The lobby-like space of K12 E. coli is reached by pass-
ing through the outer membrane. This ‘lobbyis
called the intermembrane space (Figure 3-16). and
it exists between the outer cell membrane and the
inner cell membrane. It is also a ‘security checkpoint’
since most molecules that enter or exit the cell must
go through this space. In the inter-membrane space,
there’s an additional physical barrier called the pepti-
doglycan. The peptidoglycan is a mesh-like protective
barrier between the inner and outer membranes of E.
coli cells (Figure 3-21 and Figure 3-23). The chemical
environment of intermembrane space (salt concen-
trations, water molecules, and other cellular mole-
cules) is more similar to the inside of the cell than to
the outer environment. In other words, the intermem-
brane space is a transitional space that helps to main-
tain a controlled environment inside the cell.
Figure 3-23. Factory lobby vs. the intermembrane space of
a cell. At your back is the outer brick wall and outer mem-
brane. You can see the inner wall/inner membrane space
just beyond the lobby.
Factory Floor
Inner lipid bilayer
Peptidoglycan
Security
Protein
Figure 3-22. There are many kinds of lipids that make up the
inner and outer membrane of E. coli cells. There is a general
structure to these lipids, and that includes an uncharged hy-
drophobic tail and a charged head group. As in Table 1-1 in
Chapter 1, the tail groups prefer to interact with each other,
while the head groups prefer to interact with one another and
the environment because environments on earth generally
have lots of water. These chemical bonding preferences lead
to lipid bilayers as well as micelles that you saw in Chapter 1.
Anatomy of a Lipid
The Building Blocks of Membranes
O
HC
H
O
O
O
P
O
C
C
C
HH
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
H
C
H
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
H
H
H
O
C
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
C
HH
H
H C
OO
-
R
Book _genetic engineering hero-AUG2021.indb 74Book _genetic engineering hero-AUG2021.indb 74 8/18/21 12:03 PM8/18/21 12:03 PM
Zero to Genetic Engineering Hero - Chapter 3 - Growing E. coli Cells 75
The Inner Walls (D)
Have a look around this beautiful lobby - we can very
clearly see the outer wall we just came through. We
can also see a number of inner walls and passageways.
These inner factory walls also play a very important
role. Some parts of the factory may need to be warmer,
colder or more humid than the lobby. These inner
walls and passageways help to regulate the condi-
tions in these different areas while contributing to
the structural integrity of the building. (Figure 3-24).
The inner wall of an E. coli cell is the inner membrane,
also made of a lipid bilayer (Figure 3-24). Just as you
saw with the outer membrane, the inner membrane
is comprised of a variety of lipids that have ‘hydro-
philic heads’ and ‘hydrophobic tails’. Just as the slime
layer is anchored to the outer membrane, the inner
membrane also serves to anchor proteins and sugars.
There are many different kinds of lipids that the cell
makes in order to create and maintain its protective
membranes: phospholipids, sphingolipids, bolalipids,
and lipid A, to name a few. All of the lipids and proteins
that make up the inner cell membrane contribute to
its structural integrity and help to encapsulate and
protect the next stop in our tour, the factory oor.
The Factory Floor (E)
Let’s pass through the doors from the lobby and down
the hall to the factory oor (Figure 3-16).
This is where it all happens! Look around, you’ll see
shiny machines humming while raw materials are
processed and rened into usable products. 3D print-
ers are reading factory blueprints to make a variety
of valuable objects. Quality control machines scan
items to ensure the size, weight, and composition are
correct before releasing them. There are even small
autonomous robots buzzing around, transporting
items in the right place at the right time.
You probably noticed as soon as you entered - it is
quite warm in here. The temperature has to be reliably
maintained at 37˚C. If you increase the temperature
above 40˚C or below 35˚C, these beautiful machines
start to shut down and make mistakes.
If you look closely, you’ll notice that this factory
is actually making and assembling the structural
components of the factory itself, including the
machinery! Wow, this is a factory of the future, a
factory that creates new versions of itself! Amazing!
That is what it is like to be in the cytoplasm of a K12
E. coli cell! All of the machinery you see are proteins.
Let’s learn about proteins, the cellular machinery that
makes the miracle we call life possible.
Figure 3-25. The factory oor and the cell cytoplasm con-
tain the machinery and blueprints necessary to “run” the
micro/factory.
Peptidoglycan Going Deeper 3-6
Peptidoglycan is made of sugars (N-acetylglucosamine) that are connected to short chains of amino acids
(alanine, glutamine, lysine, glycine) to form the interconnected mesh. As you now know, cells can create
hybrid macromolecules. These are a combination of different macromolecules, for example, a sugar bound
to a lipid (to make LPS).
Peptidoglycan is a mix of sugars and amino acids, and you’ll learn about amino acids soon. Over billions
of years, cells have been doing ‘molecular mash-ups’ to nd new and interesting molecules that help them
survive. What is really cool, is that they are almost exclusively made up of CHOPNS!
Figure 3-24. Factory inner wall vs. the inner membrane of a
cell. You have passed through the lobby.
Factory Floor
Inner lipid
bilayer
Protein
Book _genetic engineering hero-AUG2021.indb 75Book _genetic engineering hero-AUG2021.indb 75 8/18/21 12:03 PM8/18/21 12:03 PM
..................Content has been hidden....................

You can't read the all page of ebook, please click here login for view all page.
Reset