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Young Scholar Program for Biology of the 21 st Century –
A Summer Course for Secondary School Teachers & Students
Yr. 2003 Program
Genetic Engineering
Aim
This experiment is aimed to insert a gene coded for green florescent from jellyfish into bacterial
cells.
Introduction
Genetic materials direct many cellular functions. The 4 nucleotides (bases): namely A, T, G and
C, arrange in different orders, and some of these nucleotide sequences called genes encode for
proteins including enzymes catalyzing various metabolic pathways.
Genetic diversity exists in a population of individuals of the same species. This is usually in
terms of differences in nucleotide sequences termed alleles of the gene. Some of these alleles
may be transcribed (transform genetic information from DNA into RNA) and then translated (transform
genetic information from RNA into amino acid sequences in a polypeptide)
into enzymes of different amino
acid sequences such that the encoded enzymes show different activities.
Biodiversity exists in another level as species diversity in which some genes are conserved and
exist in different species while some genes are not present in all species. For instance,
autofluorescence can be a defence mechanism to frighten away enemies or a lightning system in
darkness. Yet autoflourescence is not needed for fierce animals, e.g. lions and tigers. A species
preserves its integrity by mechanisms to actively destroy any non-self invaders. Bacteria
biosynthesize restriction enzymes (限制酶，限制性內切酶) to cleave DNA molecules which
have recognizable sequences not owned by themselves. As a result, genetic recombination (基因
重組) to generate diversity for adapting the fluctuating environment is only restricted to members
of the same species.
Genetic engineering, in the most common format, is recombinant DNA technology (also called
gene cloning, DNA cloning), in which we use artificial means to do recombination. Through this
artificial means, an organism acquires a trait.
DNA cloning requires:
Young Scholar Program for Biology of the 21 st Century –
A Summer Course for Secondary School Teachers & Students
Yr. 2003 Program
1. target DNA fragment
2. vector to carry the DNA.
3. host cell to house the recombinant vector carrying the foreign DNA.
In order to create a recombinant DNA molecule with a target gene ligated to the
vector arm, restriction enzyme is added to the genome (基因組; the total genetic content of a cell)
of a cell, which is the source of the target gene, and to the circular vector DNA molecules. After
digestion, free single strand end regions are created (recall that DNA is a double strand molecule)
from the target gene and the vector.
The circular vector DNA is then linearized and thus
called vector arm. Ligase is added to catalyze the joining
of two strands of DNA between the 5’-phosphate and the
3’-hydroxyl groups of adjacent nucleotides. Thus a
recombinant DNA molecule is created. Vectors to be
used can be bacterial plasmids (質粒), bacteriophage,
yeast artificial chromosomes and others and are usually
chosen based on the size of the target gene. For both the
target gene and the vector DNA, extraction and
purification are needed as a preparative stage. This is called
DNA preparation.
Young Scholar Program for Biology of the 21 st Century –
A Summer Course for Secondary School Teachers & Students
Yr. 2003 Program
Transformation (genetic transformation) is a process
describing a host cell takes up an exogenous DNA /
gene. If the host cell can express the introduced gene, it
will then acquire a new trait. As ligation, transformation
and the whole DNA cloning process may not be
completely efficient and always succeed, a screening
and selection procedure is usually implemented to select the transformed host cell carrying
desirable recombinant DNA molecules. Screening is to detect for the transformants which are
the host cells harbouring DNA vectors while selection is to enable an experimenter to isolate the
transformant which is also a recombinant (a cell carries a recombinant DNA molecule). By in
vitro cultivation, the recombinant cell can be mass produced and then preserved as a clone (複
製，克隆. mass of cells of identical genetic makeup).
Genetic engineering is, sometimes, a substitute of breeding in which the desirable trait is obtained
by genetic crossing, screening and selection. Gene cloning, of course, can be carried out between
two individuals of the same or different species.
Transformation, Screening & Selection for Recombinants
a. Conservation of Gene Functions among Lives
Problem: Can a eukaryotic gene express in a prokaryote?
Propose a Hypothesis: The cloned eukaryotic gene expresses in a bacterial host.
Test of the Hypothesis: Transform a jellyfish gene (encoding green fluorescence
protein) and express it in E. coli (大腸桿菌)
Decision Making: Reject the hypothesis if the result of the test fails. This is the general
procedure of a scientific investigation.
b. Creation of life
Aim: to create an organism (bacterium, fish, fungus, etc.) producing green fluorescence.
In this case, no hypothesis is set. It is just an application of recombinant DNA technology.
The success of the experiment lies in the creation of a novel organism.
In this laboratory, we carry out a scientific investigation on the conservation of gene
functions across kingdoms .
Young Scholar Program for Biology of the 21 st Century –
A Summer Course for Secondary School Teachers & Students
Yr. 2003 Program
Background
a. The pGLO Plasmid (質粒. a circular strand of DNA) mainly consists of:
i. Target gene coding for a bioluminescent green fluorescent protein (GFP) of jellyfish Aequorea
victoria. The protein when illuminated under ultraviolet light shows a green fluorescence. Thus
expressing host cells producing GFP also show green fluorescence under UV illumination. A
marker for selection of recombinants.
ii. Antibiotic resistance gene: codes for beta-lactamase which inactivates ampicillin. This gene is
carried by the vector plasmid and not the host cell. A marker for screening for transformants.
b. Host: E. coli strain HB101.
This host is susceptible to the antibiotic ampicillin. A transformant, however, acquires resistance
against the antibiotic by possessing the plasmid carrying the beta-lactamase gene. A mechanism
of antibiotic resistance.
1. Selection and Screening for Clones having the GFP-encoding gene
Survivors on plates supplemented with ampillicin are plasmid-containing bacterial cells. They are
the transformants.
Use plates supplemented with antibiotic ampicillin and substrate arabinose (called the selection
medium) to select for recombinants (Clones having the GFP-encoding gene and express green
fluorescence under UV illumination).
Overview:
 Mixing of Plasmid DNA and host cells.
 Transformation by Heat shock.
 Enrichment of the transformants by growing in nutrient broth with antibiotic. Cells are
allowed to grow and produce beta-lactamase.
 Spread of the transformation suspension to different plates. Incubation at 37C overnight.
 Selection for recombinants as detected by autofluorescence under UV illumination.
Precautions :
Aseptic technique. Careful operation to avoid spillage.
Young Scholar Program for Biology of the 21 st Century –
A Summer Course for Secondary School Teachers & Students
Yr. 2003 Program
Be extremely carefully with ethanol and flame.
Avoid direct exposure to ultraviolet light.
Procedures:
1. Mixing of Plasmid DNA and Bacterial cells:
2.
-
Add 5 l (= 5/1000 ml) plasmid solution to the bacterial cells labeled as +DNA.
-
Add 5 l sterile water to the bacterial cells labeled as –DNA (negative control).
-
Incubate at ice for 5 min.
-
Label the LB plate (nutrient rich medium): -DNA.
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Label one LBA (LB + ampicillin): +DNA;
-
Label the LBAA (LB + ampicillin + ararbinose) plate +DNA;
-
other LBA plate: -DNA.
3. Transformation by Heat Shock: Using foam rack as a holder, transfer both (+) and (-) tube
into the WATER BATH set at 42C for exactly 1 min. After heat shock, put the tubes into ice
immediately and incubate for 2 min.
4. Development of Antibiotic resistance: Add 350 l LB broth to the each tube and incubate the
tubes at room temperatures for 10 min.
5. Spreading Plate: Pipette 100l of the transformation and control suspension onto the
appropriate plates. Spread the suspension with a L-Shaped spreader (home-made).
6. Incubation: Place the plates upside down in a 37C incubator until next day.
7. Detection: Illuminate the plates under UV. Avoid direct exposure.
Discussion
1. What is the function of the negative control?
2. Why are the plate cultures incubated upside down in a 370C incubator?
3. Name one measure to avoid the direct exposure to UV.
4. Assess the efficiency of the experiment.
5. Is the hypothesis rejected? Is the statement ‘Gene functions are conserved across kingdoms’
true?
6. How to identify genetically-modified (GM) food?
Hint. The food product contains genes only present in vector but not in the natural host. (e.g. the
ampicillin- resistant gene in the plasmid which is not present in E. coli host cells).
Young Scholar Program for Biology of the 21 st Century –
A Summer Course for Secondary School Teachers & Students
Yr. 2003 Program
7. If the trait is encoded by a plasmid and not by the bacterial DNA, what is the effect on
inheritance?