HOME-BASED INDIVIDUAL ACTIVITY:

Activity No.4 STEP BY STEP

Below are the basic steps in performing GEL Electrophoresis
Arrange them in proper order by numbering them 1-5.
_____a. Load the DNA sample into the gel.
_____b. Stain the gel and analyze results.
_____c. Hook up the electric current to the gel
_____d. Set up the gel
_____e. Make the gel
BIOTECHNOLOGY 8
March 6, 8, 2024
Continuation lesson
B. VECTORS
A Cloning vector is a DNA molecule
used as a vehicle to transfer foreign
genetic material.
It is a specialized DNA that can enter a living cell and
provide means for its detection of its presence to a researcher
by conferring a selectable property on the host cell (e.g.
resistance to antibiotics), and possess means for self-
replication.
A vector must also possess a easily
distinguishable physical traits, such as size,
or shape, to allow purification away from
the host cell’s genome.
• An ideal vector should be small in size.
• It has:
- single restriction endonuclease site
- an origin of replication
- 1-2 genetic markers (to identify recipient
cells carrying vectors)
What are the most
important vectors ?
 plasmids
The
most bacteriophages
important cosmids
vectors
phasmids
Phasmids
 PLASMID Are the most
commonly
S
used cloning
vectors
make good They are
vectors easy to
manipulate Plasmid is small in size, circular in shape
and it is a piece of DNA that is not the same
or isolate as chromosomal DNA.
because they replicate in bacterial cells, independent of the control of the
chromosomal DNA
 One of the first
widely used pBR32
plasmid DNA 2
vectors.

pBR322 was designed to have genes for ampicillin and

tetracycline resistance and several useful restriction
sites.
Plasmid – Functions:

1.They carry at least 1 gene, and most of the genes are helpful
to their host organisms.

2. They increase the survival of the organism.

3. Facilitate the process of replication.

Why is Plasmid Important?

They are important for the role they play in technological breakthroughs.

1.They have played a stellar role in the development of molecular

biotechnology.

2.They act as vehicles to introduce foreign DNA into bacteria.

3. The DNA they deliver have genes for antibiotic resistance.

4. It is a therapeutic platform for treating infectious, genetic and acquired

diseases.
Resistance to antibiotics refers to the ability of
bacteria or other microorganisms to withstand
the effects of an antibiotic that was originally
effective for treating infections caused by those
microorganisms.
When bacteria develop resistance to antibiotics, it means
that the drugs are no longer as effective in killing or
inhibiting the growth of the bacteria, making it more
challenging to treat infections.
Some plasmids can make their host bacterium
resistant to an antibiotic. In this image, two halves of
an agar plate containing the antibiotic kanamycin
have been spread with the same strain of E. coli. The
bacteria spread on the right-hand side of the plate
contain a plasmid that confers resistance to
kanamycin, so they can grow colonies even when it is
present. On the left-hand side of the plate, the
bacteria lack the kanamycin resistance plasmid and
have been unable to grow.
Nomenclature
of Plasmids
 Nomenclature of plasmids:
Nomenclature, a collection of rules for naming
things, is important in science and in many other
situations.
The name “pBR322” conforms with
the standard rules for vector
nomenclature
 first letter/s of
researcher’s names
BOLIVAR & RODRIGUEZ
(researchers)
Lower case “p”
to designate plasmid pBR322 Numerical number
given by the workers

pUC is a plasmid from University of California

* Some plasmids are given names of places

 has a DNA sequence of
4, 361 bp

Most popular Regarded as the

and widely parent or
used plasmid
vector
pBR322 grandparent of
several other vectors

It carries genes resistance to ampicillin (Ampr) and tetracycline (Tetr) that

serves as markers for the identification of clones carrying plasmids has
unique recognition sites for the action of restriction endonucleases such as
EcoRI, HindIII, Bam
PLASMIDS has unique recognition sites
for the action of restriction
endonucleases such as:
a. EcoRI c. BamHI e. PstII
b. HindIII d. SalI
Recognition site :
1.The site recognized by a restriction enzyme to
cleave
DNA is called a recognition site.
2. These sites are located on a DNA molecule
containing specific sequences of nucleotide (4-8 base
pair)
Example: The restriction enzyme ECOR1 cleaves
the DNA by recognizing a 6-base pair recognition site
 These enzymes occur naturally in bacteria as a defense against
bacteriophages-viruses that infect bacteria.

It cuts the DNA double helix at a specific site.

EcoRI It is a type II restriction enzyme, which cuts specifically at

the restriction site having 5’-GAATTC-3’ sequence.
(Restriction
Enzyme/ Restriction site of EcoRI is a palindrome and it cuts DNA
after G forming sticky ends with AATT.
Restriction
Endonuclease) Derives its name from the bacterium Escherichia coli. “R” is
for the strain RY13 and “I” as it was the first enzyme to be
isolated from the given strain.

So EcoRI is pronounced as Eco-R-One.

The cutting pattern of EcoRI is-
ACTIVITY 1: VIDEO PEEK
Activity 1 : Video Peek
CLONING VECTOR
-is a DNA molecule that carry GENE of INTEREST into a
HOST ORGANISM- thereby transform that organism into a
GENETICALLY MODIFIED ORGANISM

Bacteriophages DNA and PLASMID DNA can be used as cloning

vectors.
-has the ability to replicate within the bacterial cells
- They have high copy numbers within bacterial cells.
A vector needs the following features
to enable cloning:
1. Origin of Replication (Ori)
2. Selectable marker
3. Cloning sites
 ORI – it is the DNA sequence where
DNA polymerase will come and bind and
Initiate the replication process by creating a
multiple copies of cells within the host cells.
If the vector is carrying the gene of interest, along
with the vector, the FOREIGN GENE OF INTEREST
will also get cloned- the desirable outcome.
1. Origin of Replication (Ori) –
is a sequence from where replication
starts. Any piece of DNA when linked
to this sequence can be made to
replicate within the host cell.
 ORI – it is the DNA sequence where
DNA polymerase will come and bind and
Initiate the replication process by creating a
multiple copies of cells within the host cells.
If the vector is carrying the gene of interest, along
with the vector, the FOREIGN GENE OF INTEREST
will also get cloned- the desirable outcome.
(Ori) – Linking a piece of DNA to this sequence
causes it to replicate in the host cell. (host cell - a living
cell invaded by or capable of being invaded by an
infectious agent (as a bacterium or a virus- pathogen)
This sequence also controls the copy number of the linked
DNA. Therefore, the target DNA needs to be cloned into a
vector whose “ori” supports a high copy number, to
recover large amounts of DNA.
2.Selectable marker – it helps in
identifying and eliminating untransformed
cells, (Non-transformed E. coli cells do not naturally have an
ampicillin resistance gene and they have non taken up the cloning
plasmid, therefore they will be unable to grow in the presence of
ampicillin.)
 Transformants non-transformants.
-the bacterial cells -the bacterial cells that
that take up the do not take foreign
foreign DNA are DNA are called non-
transformants.
called transformants,
The transformants are distinguished from non-
transformants with the help of antibiotic-resistant genes in
the cloning vector.
Recombinant DNA refers to a piece of DNA which
combines with another foreign DNA to form a new DNA
molecule.

Non-recombinant DNA refers to the parental DNA or

original DNA which does not contain any foreign DNA
Selectable marker – The vector needs to have a
selectable marker that allows the selection of recombinants
over non-recombinants.
In terms of E. coli, some useful selectable markers are
genes that provide resistance to antibiotics:
- ampicillin
- kanamycin
- chloramphenicol
Since normal E. coli cells do not carry these resistance
genes, it becomes easy to select the recombinants .
RECOMBINANT DNA (rDNA)

-Refers to a piece of DNA which combines with another

foreign DNA to form a new DNA molecule
3.Cloning Sites - it is a place where the gene of
interest will attach.
To attach the foreign DNA to a vector , the vector should have a
recognition sites for a specific restriction enzyme.

Multiple recognition sites will result in a multiple DNA

fragments, complicating the process of cloning.
A vector has a more than one antibiotic resistance gene. The
foreign DNA is ligated into a restriction site in one of the
antibiotic resistance genes.
c. COMPETENT HOST
Host cells – are bacterial cells that take up
Recombinant DNA.
- Since DNA is hydrophilic, it cannot pass
through the cell membrane of bacteria
easily.
Therefore, the bacterial cells have to be
made “competent” to take up the DNA.
A competent host is referred to as the
ability of bacterial cells to become the
host for foreign DNA.
Competent Host Cells
Plant Animal
cells cells
 The following are the different techniques to make
host competent:

1. CHEMICAL TREATMENT
●Treating hosts (bacteria) with a specific concentration of a divalent cation
increases efficiency and make DNA enter through the cell wall of the host.
● Heat shock (sudden change in temperature
● Incubation of host and rDNA on ice and then placing them at 42⁰C
● Enable bacteria to take up rDNA.
2. MICROINJECTION
● rDNA directly injected into the nucleus of the host cell.
● common for animal cell
 The following are the different techniques to make
host competent:

3. BIOLISTIC AND GENE GUN

●The host cell is bombarded with high velocity micro-particles of
gold or tungsten coated with DNA.
● used for plant cell

4. DISARMING PATHOGEN
● Disarmed pathogen vector-allowed to infect host & transfer rDNA.
MINI LAB ACTIVITY NO. 4
CLONING VECTOR
Objective: To be able to show how the different features
for cloning vectors are utilized to create rDNA.

Materials: Coloured paper cut out

pentel pen
scissor
paste and long coupon bond
PROCEDURE:

1.Use the pBR322 (DNA cloning vector) diagram as point of reference.

2.Cut out colored papers to form the diagram with corresponding shape
to represent the different features in cloning.
4. Label your diagram – using all the features needed in cloning such as:
origin of replication (ori), selectable markers: ampicillin, tetracycline
recognition sites,/cloning sites, EcoRI, gene of interest, etc. )
5. Tell the process how these features in cloning can help the process its
process.
6. Answer the questions that follow.
 Check your understanding:
1.What makes up an ideal vector?
2. Why are plasmids the most commonly used
cloning vector?
3. How do plasmid vectors acquire their specific
name?
4. What are the essential elements of a plasmid cloning
vector?
5. Can plasmids replicate?
 Check your understanding:
6. Is it desirable to have multiple cuts in multiple
recognition sites? Why or Why not?
7. How can cloned vectors help defend against
pathogenic invaders.

8. When do you say that the host organism is resistant

to antibiotics.