Chapter 2

Recombinant DNA technology

Important dates
 Karl Ereky coins the term biotechnology, present for more than a hundred
years = not a new concept.
 1953 - Watson and Crick determined the structure of DNA
 1970s-discovery on restriction endonuclease (EcoR1)
 1990 - approval granted in the US for a trial of human somatic cell gene
therapy - it was illegal to manipulate humans with genetic mutations.
 2001 - Human genome sequence is published
 CRISPR technology - NIH approval to test its safety in a clinical trial - Big
controversy because of the harm it can cause but for certain diseases it was
proven to be successful
Steps in genetic engineering
1. Prepare the gene
2. Cut the DNA into fragments using restriction enzymes.
3. Cut the vector & insert gene into vector
4. Choose appropriate host cell
5. Host transformation
6. Selecting recombinants

1. Prepare the gene

Purify the chromosomal DNA from proteins, RNA (using enzymes, proteases,
RNAse, etc.)
Also purify using phenol chloroform extraction
We can test the purity of a sample by running optical density measurements or
spectrophotometry (260-280nm).

2. Cut the DNA into fragments using restriction enzymes.

 Discovery of restriction endonucleases
They were discovered when we found out that certain bacteria are resistant to
infection by phage (similar to Hershey & Chase experiment).
Experiment: some bacteria did not lyse after being
exposed to bacteriophage (no plaques on the agar
plate) and they found enzymes inside the bacterial
cells that protect the bacteria by fragmenting the viral
DNA and inhibiting the replication of the phage.
  Types of restriction enzymes
Type I and III: Not favorable
- Cut 1000 nucleotides from restriction site
- Methylase and nuclease activities in one enzyme: the enzyme would
unpredictably either methylate DNA or cut DNA.
- Need ATP for enzyme activity= energy-costly

Type II: Favorable

- Cut at the recognition sequence
- Methylase and nuclease activities are separate: the nuclease part can
be bought alone and only cuts the DNA.
- Need Mg as co-factor (cheaper than ATP)

 Naming the enzyme

Each type of endonuclease came from a bacterial cell, therefore:
- 1st letter: genus of the bacteria
- 2nd two letters: species of the bacteria
- Strain of the bacteria (if there is)
- Order of discovery
Example: BamH1 (Bacillus, amyoliquefaciens, strain H, 1st one discovered in that
strain).
Why don’t restriction enzymes digest chromosomal DNA in bacterial cells?
Because bacterial chromosomal DNA is methylated.
  Properties of Type II
- Short recognition sequences: 2,4,6,8 nucleotides.
- Palindromic: symmetry
- Types of ends: sticky (overhang), blunt, or incompatible cohesive.

Sticky end enzymes: Eco R1, BamH1

Advantage: We can use the same enzyme to cut the vector DNA and insert DNA,
and then to retrieve the DNA insert from the plasmid vector (easy to anneal=no
extra energy needed)
Once the insert is put, nicks are generated. They can be sealed using DNA ligase,
which is an ATP driven molecule: when the ATP breaks down into ADP + Pi, it
gives a phosphodiester bond to ligate the DNA.

Disadvantage: the vector could re-anneal

on itself without integrating the DNA insert.
To prevent this, we add alkaline phosphatase in the test tube along with the
vector and the restriction endonuclease. This generates hydroxyl groups on both
ends (breaks PO4- bonds), making it unlikely for the vector to anneal on its own.
Note: we only add small concentrations of alkaline phosphatase that will be
consumed in the reaction.

2 of the 4 nicks are sealed, joining the 2

DNA molecules covalently.

Blunt end enzymes: Hae III

Advantage: used in making Polylinkers (multiple cloning sites-mcs).
Polylinkers are sequences of recognition sites of different enzymes that are put
one next to each other in a tandem array using blunt end ligation. That way,
researchers can use any enzyme they want in a flexible way to conduct their
experiments.

Disadvantage: more difficulty in annealing, that’s why we

increase the concentration of DNA ligase in the mixture.

Incompatible cohesive termini: HindIII and XbaI

 These are overhangs that are not completely sticky and compatible, because of
the nature of the cut.
Disadvantage: we have to fill in the missing nucleotides to make them completely
sticky and compatible, by adding dATP or dGTP generate sticky ends.

3. Cut the vector and insert gene into vector

Vector type Maximum Applications Limitations
insert Size
Plasmid vector 6-12 kb -DNA cloning -Restricted insert size
(circular) -Protein expression -limited expression of proteins
-subcloning because bacteria don’t have
-direct sequencing of insert transcriptional enzymes/factors
DNA -copy number problems
-only host: bacteria/yeast (≈ )
Bacteriophage 25 kb -cDNA, genomic and -Packaging limits DNA insert size
vectors (linear) expression libraries -host replication problems: bacteria
only
Cosmid (circular) 35 kb -cDNA and genomic libraries -Phage packaging restrictions.
-cloning large DNA -not ideal for protein expression.
fragments -cannot replicate in mammalian cells
Vector type Maximum Applications Limitations
insert Size
Bacterial artificial 300 kb -Genomic libraries -only host: bacteria since they
chromosome -cloning large DNA originate from bacteria
(circular) fragments. -cannot be used for complex protein
expression; only for simple (ex:
insulin in E.coli)
Yeast artificial 200-1000 -Genomic libraries -only host: yeast
chromosome kb -cloning large DNA
(circular) fragments.

Ti vector (circular) Varies -Gene transfer in plants. -only host: plants

=tumor inducing depending -number of restriction sites randomly
on type of distributed
Ti vector -large size of vector not easily
manipulated.

 Copy number problems

- High copy number plasmids: the host allows it to replicate as many
times as possible
- Low copy number plasmids: restricted to replicate once or twice
within the host
The copy number restriction is a sequence that is usually removed for
manipulation.

 What determines the choice of vector?

- Insert size (should match the vector)
- Vector size (should match the insert)
- Restriction sites and endonucleases available
- Copy number (how much yield we want to produce)
- Cloning efficiency: efficiency of transformation (how many of plasmid
went into the host).
- Ability to screen for inserts

 Types of vectors
Plasmids
Why do bacteria have plasmids? Different types of plasmids exist naturally to
provide bacteria with resistance to antibiotics.
Definition: they are self-replicating, extrachromosomal pieces of DNA.

High copy nbr (no control, relaxed hosts) [100-500 copies/cell].

Low copy nbr (restrictive hosts/stringent control) [<10 copies/cell].
To increase yield= use relaxed host and remove the genes from plasmid.
To decrease yield= use stringent host and keep the genes (we use it when the
product is toxic to the cell).
Properties:
- Replicon/origin of replication (host specific): the site to which DNA
pol attaches to initiate replication
- Selectable marker (usually an antibiotic resistance gene)
- Unique restriction sites: the site is not repeated twice because this
could cut the plasmid many times and some pieces will be lost.
- Remove mobilization (mob) genes that allow the plasmid to move
between bacterial cells through conjugation.
Because during this process, the plasmid could be modified and
ruined.
Examples of plasmids
- pBR322 (earliest): E.coli replicon// Amp AND Tet resistance genes
(used as selectable markers)

total size: 4kb

To screen for the recombinant plasmid vectors:
Insertional inactivation: if I use a restriction endonuclease whose site is within an
antibiotic resistance gene to insert my gene of interest, then the antibiotic
resistance gene will be inactivated.
Replica plating: from an initial master plate, transfer each colony to two identical
plates in an identical way, with a sterile toothpick. Then, we culture each of those
two plates in media containing tetracycline or ampicillin. The colonies that
survived and can be recovered are used for further analysis.
For example, if we inserted the gene of interest using an endonuclease that
disrupts the tet resistance gene, we culture it on tetracyclin media and those who
die are the recombinants.
 - pHV33: E.coli and B.subtilis replicon//Amp, Tet, Chl resistance.

-oriE: origin of replication for E.coli

-oriB: origin of replication for B.subtilis

- YRP 17: yeast (ars) and E.coli// amp, tet resistance

-trp 1: synthesis of tryptophan

-ura 3: synthesis of uracil

- pUC plasmids: modified pBR322

most widely used (replaced others)
Select in a histochemical way (using colors), instead of replica plating and
antibiotic resistance.
tet resistance genes removed
copy nbr genes removed (higher yield)
 polylinker sites added (mcs)
 LacZ (codes for beta-galactosidase) and LacI (codes for a repressor that blocks
the transcription of lac Z gene) genes added [taken from the inducible lac operon
that allows bacteria to survive with lactose].
Total size: 2Kb

For selection:
Xgal+ beta galactosidase  blue product (white colonies= insert)
IPTG: inducer of the lac operon (blocks the Lac I repressor).
Phages as vectors
They can transfect animal cells (except bacteriophages) but only replicate in
bacteria.
Larger DNA insert than plasmids, but we are limited to a total of 50 Kb due to
packaging restrictions (phage genome+ insert together)
What is packaging? inserting a piece of DNA into head of the phage (protein coat), at the later
stage of infection (when the phage is about to leave the cell to proliferate). Packaging
restrictions imply that DNA cannot be larger than a certain size

Note: adenoviruses; HIV; retroviruses can be used for animal cells.

Bacteriophages can be “Filamentous phages”: single stranded DNA phages that
infect E.coli and can be recovered either as single stranded (phage) or as a double
stranded (plasmid). (Ex: phage M13).

Bacteriophage M13
It has 2 forms:
- filamentous, rod-shaped, with single stranded circular DNA (useful in
sequencing and mutagenesis)
- Replicated form (RD): double stranded circular DNA.
In the process of infection, M13 injects its ss circular DNA gene into the bacterial
cell. Inside the bacteria, the injected DNA is replicated to form replicated forms.
We can perform a simple plasmid preparation to get RF of phage DNA by lysing
bacterial cells using detergents. After that, we can clone it by using it and cut it
with restriction enzymes.
We could also wait for injection to happen, and isolate new phage particles that
are back to single stranded form (we streak the plate and select the plaques from
which we take ss phage DNA).
A section in M13 is not required for the life cycle and is called intergenic space. It
can be removed to add foreign DNA fragments (inserts of interest) at the place.

Side note: Lytic VS lysogenic life cycle

- Lysogenic: the phage attaches, injects the DNA into the bacterial cell,
and DNA gets incorporated into bacterial cell genome changing its
properties, but it doesn't lyse. The phage DNA that is incorporated
within the bacterial DNA is called a prophage, then it continues to
replicate.
- Lytic: the phage attaches, injects the DNA into the bacterial cell, uses
the bacteria's machinery to assemble new phage parts and lyse the
cell before propagating into others.
Transparent plaques in agar plates are the phage particles that lysed
bacterial cells.
This is the single stranded version of the DNA
Phage lambda
double stranded linear DNA
whole genome is 50Kb
we can use modified versions of the phage whose lysis can be induced by
temperature variation in the medium.
Also subject to packaging restrictions
Used for cDNA and genomic libraries

Phage lambda has 2 cos sites (short sequences) at each of both ends of its linear
genome, one on each of the two strands (4 cos sites in total).
When phage lambda replicates, it is in a linear fashion, but the phage genome
circularizes upon infecting a host.
This is due to an enzyme in the phage head that recognizes the cos sites and
breaks one of the two on each end. That way, we are left with only one cos site on
each strand.
They are complementary to each other, so they stick to each other and that is
how the genome circularizes upon infection (in the head it is linear since no space
to circularize)

Since the genome is now

temporarily circular, the bacteria will see lambda
as a plasmid. The phage can now replicate itself
and then be packaged into the protein coat of the
new phages being assembled.
Phage lambda contains a 20 kb-long fragment in
the genome that is not needed for the life cycle. A
BamH1 site spans both ends of this fragment, thus
we can remove the fragment completely by using
BamH1 and insert a 20-kb fragment instead.
To select only the vectors that integrated the insert of interest and not the ones
that annealed on themselves, we use P2-E.coli, that allows only the recombinant
phages to grow and form plaques.
Cosmids
They are man-made vectors, unlike plasmids and phages.
They are made by taking cos sites (ends of phage lambda genome) and circularize
inside the host like plasmids.
Properties:
- Cos sites from phage lambda, and everything else can be our inserts.
- Replicate as plasmids once they infect a bacterial host.
- Same packaging restrictions as phage lambda (max 50 kb)
How to use cosmids?

1. Linearize plasmid by using restriction enzymes (Sca1, for example), cos sites will
be flanking both ends
2. BAMH1 cut to generate 2 fragments (with sticky ends).
3. Take sequence of DNA and use restriction BAMH1 to generate fragments
4. Mix both the plasmid and the DNA fragments
5. Anneal using DNA ligase.
Packaging takes place naturally in vitro to
generate phage particles that contain cos
sites from phage and the DNA insert.
After infection of E.coli, the piece of DNA
circularizes (because cos sites are single
stranded and complementary) and
replicates as a plasmid. That is when the
origin of replication becomes important.
Yeast artificial chromosome (YAC) [shuttle vector]
It has telomeres to protect DNA from degradation; + ori site+ centromere.
It was used to clone large DNA fragments of human genome (100-500kb).
To sequence human genome, it was cut with restriction enzymes that have CG
rick recognition sequences (ex: NotI, SmaI) because these are not frequent in the
human genome. These are called mega base enzymes since they generate mega
size fragmented genome.
We wanted to generate large fragments to
have less number of colonies to analyze.

Partial digest was also used: reduce the

concentration of endonuclease and time of incubation of the enzyme to generate
large size fragments.
TI vector (tumor inducing plasmid)
Comes from a bacteria that infects plant cells and injects its DNA in them to
produce cancer (genus: agrobacterium).
This bacterial genomes is used as a vector by removing the genes that cause
tumor formation (code for plant hormones).

4. Choose the appropriate host cell

The host cell is chosen based on the product yield, the growth rate, the activity of
the product (how complex the product is; use in euk or prok??) …

5. DNA transfer/transfect to host

DNA uptake by E.coli or yeast  transformation
DNA uptake by animal/plant cell  transfection

Transformation of E.coli
-Easy to culture
-Replicates quickly: once/22 min  11 hrs gives 1 billion cells
-Have an easy media to thrive: LB broth (yeast extract, NaCl, tryptone, pH 7.5 …)
-Oxygen: continuous shaking on small scale or providing bioreactor with gas on a
large scale.
-Phases of growth: Lag (adapt to medium), Log, Stationary
phase, death. We transform them while in the log phase.
-Transformation efficiency: 109 transformants per microgram
of DNA.
-Aseptic technique required.
-Use competent E.coli (that can uptake DNA fast). They are in the log phase cells -
they are adhesive in that phase because there is a fusion between the outer and
inner membranes of E.coli, generating "pores" in the E.coli membranes which
serve as adhesion zones for DNA uptake.

-Use of CaCl2/ice to speed up the transfer to cell

membrane and coat the DNA. Ca2+ complex with
negatively charged DNA to shield DNA phosphates (-) from
being repelled by the phospholipids (-) at the adhesion
zone.

Steps of transformation
1.Streak plate/grow bacterial colonies
2.Pick colony/grow bacteria in broth (we use
spectrophotometry to test the optical density of cells: 108
cells/mL  cells are in log phase)
3.Incubate cells in ice in 0.1M CaCl2
4.Centrifuge and obtain cells (pellet)
5.Add DNA
6.Heat shock cells (42C) for 90s (to obtain more transient
pore in membrane of E.coli to uptake DNA). Sometimes, we
use Turbo Cells (really competent E.coli) that just need a heat shock at 37 degrees
for one minute. This is less costly and more effective because there is no need for
cooling (less time consuming).
7.cool sample (with ice) for antibiotic resistance recovery (since proteins die at
42C)
8.Streak again and select recombinant colonies.
Transfection to mammalian host
Direct (vector-less) gene transfer:
- Calcium phosphate co-precipitation
- Electroporation
- Lipofection
The transfection can be:
- Transient: short-lived
▪The gene gets into the cytoplasm, crosses the nuclear membrane, gets expressed
in the nucleus, and then is lost in the second generation no incorporation in the
genome of the cell.
▪Used to study gene expression  we can see results faster
▪We can look at the expression of a gene for 72 hrs MAX.
▪Involves reporter genes (reports the expression level of the gene):
- luciferase (LUX or LUC): isolated from the firefly/bioluminescent
bacteria  produce light in the presence of luciferin (measure light
using luminometer) (ex: Vibrio fisheri bacteria)
We place the reporter next to the gene of interest
- Green fluorescent protein (GFP).

- Stable: long-lasting
▪The gene crosses the cell membrane, gets into the nucleus, incorporates in the
genome, and then transfers from generation to generation.
▪It involves the selectable marker: nptII (Neomycin phosphotransferase-related
gene [bacterial resistant gene modified for mammalian cells]) provides resistance
to G418 drug, which stops DNA synthesis (blocks S phase).
We can select for cells by using G418: This selection is done for 7-10 days to make
sure that only those who took the gene are dividing.
Optimizing G418 concentration
If the concentration is too high, the cells will die even if they have the resistance
gene (npt2)  we need to find a concentration that is optimal to the living of the
cells at a certain density (if the cell nbr is very low, even a small concentration
could kill the cells)

Selection of stable transfectants:

▪3 techniques can be used to generate both transient OR stable transfections:

 Electroporation Lipofection Calcium-phosphate
coprecipitation
Culture cells + DNA that we wish to Some lipid is being used: lipid Calcium-phosphate
insert into cells solution bought from precipitates with DNA.
company (Lipofectamine = a
Mix Cells and DNA in a special lipid bilayer). Use of adenoviral DNA
cuvette (electroporator) particles, add phosphate buffer
Apply voltage which creates It is mixed with DNA, and to it and CaCl3 (generate
transient pores in the cells, naturally, the lipid bilayer Calcium phosphate DNA
allowing the DNA to enter fast. "encases" the DNA in a precipitates).
"liposome." [=protection]. This precipitate is important
Apply selection to get stable Liposomes adhere to cells and because cells have a large
expression OR no selection to get go inside them need for calcium.
transient expression
Advantage of liposome: They Once calcium is taken, DNA is
Things to optimize: dissolve easily in the taken with it because it has co-
-Amount of voltage: Too high/much cytoplasmic membrane, precipitated with the
electricity can kill the cells and that is how the DNA is phosphate.
-Duration of pulses: Voltage must delivered:
be pulsed onto cells (not constant) Cytoplasm then Nucleus Add CaCl3-DNA co-precipitate
-Number of pulses: Pulses must be + wait some time.
limited (too many might kill the Gene is expressed
cell) This is transient or stable. For transient: leave tray for a
 few days.
Cubosomes: cubes with lipids For stable: more time
in them

6. Selection of recombinants (after transformation)

a. Nutritional complementation
We choose a mutant E.coli that cannot grow without a certain amino acid being
supplemented in the medium (ex: trp-)
We supplement the gene for that amino acid within the insert  only the cells
that grow in an amino acid negative medium are the ones that have taken up the
insert.
Note: this technique is not evident since the vector could reanneal on its own
while having the amino acid gene but not the insert.
We complement the missing gene in some yeasts we buy (that are deficient ), by
inserting the gene in our insert [not in the vector]

b. Antibiotic resistance (ex: ampicillin)

Ampicillin has a beta-lactam ring that mimics a protein cross bridge.
Ampicillin works by incorporating that beta-lactam ring in the cell wall of E.coli
during cell wall biosynthesis (beta-lactam blocks cell wall production). Therefore,
there will be no cross linking for the protein and the cell will die.
Ampicillin resistance genes code for beta-lactamase, which cleaves the beta-
lactam ring of ampicillin the antibiotic resistance gene codes for an enzyme
that deactivates it.

c. Detect presence of product if it can be assayed: insulin, growth

hormone…
Not widely used because it is cumbersome
d. Histochemical methods of selection
It is based on color formation
Ex: LacZ (white cells  transformed)
e. Agarose gel electrophoresis
Steps:
 Prepare plasmid DNA
1.Isolate plasmid DNA from complex chromosomal DNA (bulky and dense), by
suspending the cells in a buffer with EDTA (chelating agent that weakens the
plasmid membrane to avoid using a lot of SDS)+ glucose (provide healthy cells)
2.Lyse the cells (use SDS to solubilize the membrane)
3.Add NaOH to denature DNA (chromosomal+ plasmid) [high pH+ high t].
4.Renature DNA by adding potassium acetate to neutralize NaOH. Note that the
plasmid DNA (smaller and lighter) renatures first.
5.Centrifuge and isolate plasmid DNA from supernatant.

 Cut with restriction enzymes to linearize plasmid DNA

 Run DNA of different colonies side by side on agarose gel for
electrophoresis, with a marker lane.
 Look for number of bands (for ex, if we cut with 1 RE, the plasmid that did
not take up the DNA insert will give one band of DNA, while the one who
did will give 2 bands)
Additional notes on agarose gel:
-Agarose: larger pores. Pore size is changeable by changing concentration of
agarose in gel mix. (smaller % --> bigger pore size --> bigger DNA can be
accommodated)
-Use bromophenol blue as a tracer so that bands don’t get confused with buffer.
-Stain with ethidium bromide (intercalates between DNA pieces), fluoresces
under UV.

f. In situ hybridization: colony hybridization

Absorbent filters: Whatman 541 (cheapest); nitrocellulose (widely used); Nylon
membranes (expensive// used if we need to reprobe/rehybridize the membrane).

Immunological methods (if the product is a protein)

-From master plate, transfer cells onto a nitrocellulose membrane (by taping or
using a toothpick), on which we lyse our cells using a detergent (membranes are
broken open).
-Add a primary antibody to the matrix to detect a specific protein; then wash up
any unbound primary antibody.
-Add labeled secondary antibodies; then wash away any unbound secondary
antibody.
Why don’t we label primary antibodies? We will need to label
every antibody for every protein that exists (every specie
that exists). A secondary body is versatile, therefore it is
cheaper and faster as we label less antibodies.
DNA or RNA hybridization techniques (if the product is DNA)
We use a probe: a small oligonucleotide (20 bp max) that can detect the insert.
1.The colony on the master plate is transferred on a nitrocellulose membrane.
2.Lyse the cells.
3. Denature the DNA into single strands by
incubating the membrane in NaOH with basic pH
(>13) or super high temperature (95 degrees).
4. Bind DNA to matrix by incubating
nitrocellulose membrane in a vacuum oven, to
avoid colony mixing.
5. Then, a labelled probe is added into the
matrix, and will hybridize with the insert.
6. Wash away non-hybridized probes.
If we want to detect RNA, we a little bit of NaOH
to prevent the formation of hairpin loops (double-stranded)+ incubate the
membrane with DNase to get rid of any DNA.
We can also use DNA probes to detect RNA => DNA-RNA hybridization is more
stable than DNA-DNA hybridizations. However, RNA probes’ specificity is not very
high usually => stability not as much as DNA

Note: The probe method is much better than gel electrophoresis because it is
more specific: it detects the sequence of DNA. Agarose gel electrophoresis is only
based on size, so all the fragments having the same size of insert will run to the
same location.

g. Southern blotting
If we don't want to use colony hybridization and we have used electrophoresis as
a first way to detect the gene of interest, we use the Southern blot as a second
technique (alternative to in-situ hybridization)
• Transfer bands from agarose gel and blot gel onto nitrocellulose membrane,
and then hybridize with a probe.
The difference between this and in-situ hybridization is that: This is done after
electrophoresis; thus DNA is cut up