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GENETIC ENGINEERING AND BIOTECHNOLOGY

Genetic engineering, also called genetic modification, is the direct manipulation of an organism’s genome
using biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the
transfer of genes within and across species boundaries to produce improved or novel organisms. New
DNA is obtained by either isolating or copying the genetic material of interest using molecular cloning
methods or by artificially synthesizing the DNA. A construct is usually created and used to insert this DNA
into the host organism. As well as inserting genes, the process can also be used to remove, or "knock out",
genes. The new DNA can be inserted randomly, or targeted to a specific part of the genome.

Insight Medical Publishing (2020), Genetic Engineering and Biotechnology Retrieved from:
http://www.imedpub.com/scholarly/genetic-engineering--biotechnology-journals-articles-ppts-list.php

A. RECOMBINATION IN NATURE

How does DNA recombination work? It occurs frequently in many different cell types, and it has important
implications for genomic integrity, evolution, and human disease.

DNA recombination involves the exchange of genetic material either between multiple chromosomes or
between different regions of the same chromosome. This process is generally mediated by homology;
that is, homologous regions of chromosomes line up in preparation for exchange, and some degree of
sequence identity is required. Various cases of nonhomologous recombination do exist, however

Nature Education (2014), Genetic Recombination Retrieved from:

https://www.nature.com/scitable/topicpage/genetic-recombination-514/

1. Recombination between bacterial species

When you hear the word “clone”, what do you think of?

-maybe experiments carried out in molecular biology labs. But its also true that the bacteria around you-
on your skin, in your gut, growing on your kitchen sink are “cloning” themselves all the time

Bacteria reproduce by splitting in two via binary fission. Binary fission makes clones, or genetically
identical copies, of the parent bacterium. Since the “child” bacteria are genetically identical to the
parent, binary fission doesn’t provide an opportunity for genetic diversity. This contrasts with
sexual reproduction. Still, genetic variation is key to the survival species, allowing groups to adapt
to changes in their environment by natural selection. That’s true for bacteria as well as plants and
animals. So it’s not too surprising that prokaryotes can share genes by three other mechanisms
conjugation, transformation and transduction

3 methods by which bacteria can undergo genetic recombination are as follows:

1. transformation

-occurs naturally in some species of bacteria, but it can also be induced in the lab.

-a bacterium takes up a piece of DNA floating in its environment, often DNA that’s been shed by other
bacteria. In a laboratory, the DNA may be introduced by scientists. If the DNA is in the form of a circular
DNA called plasmid, it can be copied in the receiving cell and passed on its descendant.

Why would this be important? Imagine that a harmless bacterium takes up DNA for a toxin gene from a
pathogenic (disease-causing) species of bacterium. If the receiving cell incorporates the new DNA into its
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own chromosome (which can happen by a process called homologous recombination), it too may become
pathogenic

2.transduction

-viruses that infect bacteria move short pieces of chromosomal DNA from one bacterium to another “by
accident”

Yep, even a bacteria can get a virus! The viruses that infect bacteria are called bacteriophages.
Bacteriophages, like other viruses, are the pirates of the biological world-they commander a cell’s
resource and use them to make more bacteriophages

-is the injection of foreign DNA by a bacteriophage virus into the host bacterium. Bacteriophages can
accidentally transfer genetic material from one bacterium to another in transduction. DNA is transferred
from one bacterium to another by a virus or viral vector

However, this process ca be a little sloppy, sometimes, chunks of host cell DNA get caught inside the new
bacteriophage as they are made. When one of these “defective” bacteriophages infects a cell, it transfers
the DNA, some bacteriophages chop the DNA of their host cell into pieces, making this transfer process
more likely.

Archaea, the other group of prokaryotes besides bacteria, are not infected by bacteriophages but have
their own viruses that move genetic material from one individual to another.

-DNA accidentally moved from one bacterium to another by a virus

-can also be used in the lab to introduce a foreign gene to bacterial host cell’s genome

3. conjugation

-DNA is transferred between bacteria through a tube between cells

-DNA is transferred from one bacterium to another. After the donor cell pulls itself close to the recipient
using a structure called a pilus, DNA is transferred between cells. In most cases, this DNA is form a plasmid

Donor cells typically act as donors because they have a chunk of DNA called the fertility factor or F factor.
This chunk of DNA codes for the proteins that make up the sex pilus. It also contains a special site where
DNA transfer during conjugation begins. If the F factor is transferred during conjugation, the receiving
cell turns into an F+ donor that can make its own pilus and transfer DNA TO OTHER CELLS. Here’s one
analogy: this processs is sort of like how a vampire can turn other people into vampires by biting them

Khan Academy (2020), Genetic Variation in Prokaryotes, Retrieved from:

B. Recombination Eukaryotic Species

In eukaryotic cells, which are cells with a nucleus and organelles, recombination typically occurs during
meiosis. Meiosis is a form of cell division that produces gametes, or egg and sperm cells. During the first
phase of meiosis, the homologous pairs of maternal and paternal chromosomes align. During the
alignment, the arms of the chromosomes can overlap and temporarily fuse, causing a crossover.
Crossovers result in recombination and the exchange of genetic material between the maternal and
paternal chromosomes. As a result, offspring can have different combinations of genes than their parents.
Genes that are located farther apart on the same chromosome have a greater likelihood of undergoing
recombination, which means they have a greater recombination frequency.
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Nature Education (2014), Recombination retrieved from:
https://www.nature.com/scitable/definition/recombination-226/

Homologous recombination, the exchange of genetic material between two strands of DNA that contain
long stretches of similar base sequences. Homologous recombination occurs naturally in eukaryotic
organisms, bacteria, and certain viruses and is a powerful tool in genetic engineering. In eukaryotes,
homologous recombination occurs during meiosis, playing a critical role in the repair of double-stranded
nicks in DNA and increasing genetic diversity by enabling the shuffling of genetic material during
chromosomal crossover. In bacteria, homologous recombination is a major mechanism of DNA
repair and facilitates the incorporation into DNA of genetic material received via horizontal gene
transfer and transformation. In viruses, homologous recombination helps shape viral evolution.

In genetic engineering, homologous recombination is used as a form of gene targeting, in which an

engineered mutation is introduced into a specific gene as a means of investigating the gene’s function. In
this approach, foreign DNA with a sequence similar to that of the target gene but flanked by sequences
identical to the ones upstream and downstream of the target gene’s location is introduced into a cell. The
cell recognizes the identical flanking sequences as homologues, causing target gene DNA to be swapped
with the foreign DNA sequence during replication. The exchange inactivates, or “knocks out,” the target
gene. In mice, this method is used to target specific alleles in embryonic stem cells, enabling the
production of knockout mice. Artificial genetic material similar to the target gene is introduced into
the nucleus of the embryonic stem cell, which represses the target gene by the process of homologous
recombination. With the target gene rendered inactive, scientists are able to deduce and investigate its
biological functions in the mouse.

Numerous mouse genes have been knocked out with the help of gene targeting, resulting in the
production of hundreds of different mouse models of human disorders, including cancer, diabetes,
cardiovascular diseases, and neurological disorders. Groundbreaking work on homologous recombination
in mouse stem cells was carried out by scientists Mario Capecchi, Sir Martin J. Evans, and Oliver Smithies,
who were awarded the 2007 Nobel Prize in Physiology or Medicine for their discoveries.

Encyclopedia Britannica (2020), Homologous Recombination Retrieved from:

https://www.britannica.com/science/gene

Recombinant DNA Technology

Recombinant DNA Technology

All organisms on Earth evolved from a common ancestor, so all organisms use DNA as their molecule of
heredity. At the chemical level, DNA is the same whether it is taken from a microscopic bacterium or a
blue whale. As a result, DNA from different organisms can be “cut and pasted” together, resulting in
“recombinant DNA”. The first recombinant DNA molecule was produced in 1972 by Stanford researcher
Paul Berg. Berg joined together DNA fragments from two different viruses with the help of particular
enzymes: restriction enzymes and ligase. Restriction enzymes (such as EcoR1 in the figure below) are like
“molecular scissors” that cut DNA at specific sequences. If the DNA from the different sources is cut with
the same restriction enzyme, the cut ends can be joined together and then sealed into a continuous DNA
strand by the enzyme ligase. In 1973, the first organism to contain recombinant DNA was engineered by
Herb Boyer (UCSF) and Stanley Cohen (Stanford University). Together they introduced an antibiotic
resistance gene into E.coli bacteria. Notably, they also produced bacteria that contained genes from the
toad Xenopus laevis , which showed DNA from very different species could be spliced together. Paul Berg
was awarded the 1980 Nobel Prize in Chemistry “for his fundamental studies of the biochemistry of
nucleic acids, with particular regard to recombinant-DNA”.
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Laguna State Polytechnic University – San Pablo City Campus
San Pablo City

The production of recombinant DNA involves cutting two different pieces of DNA with the same restriction
enzyme and then ligating (“glueing”) the pieces together. Image courtesy of Wikimedia Commons

The ability to cut, paste, and copy molecules of DNA was not only a watershed moment for scientific
research but spawned an entire industry built on genetic engineering. Genetech, the first biotechnology
company, was founded by Herb Boyer in 1976. By 1982, the FDA approved Genetech’s first successful
product, a synthetic form of human insulin produced by bacteria that were engineered to contain the
insulin gene.

Today recombinant DNA technology is used extensively in research laboratories worldwide to explore
myriad questions about gene structure, function, expression pattern, regulation, and much more. One
widely used application involves genetically engineering “knock-out” animals (typically mice) to contain a
non-functional form of a particular gene of interest. The goal of such experiments is to determine gene
function by analyzing the consequences of the missing gene. While knockout mice are generated to
answer questions in many different fields, they are particularly useful in developmental biology and have
led to an understanding of some of the essential genes involved in the development of an organism from
a single fertilized egg.

Recombinant DNA techniques are also a cornerstone of the biotechnology industry. One example is the
generation of genetically engineered plants to produce an insect toxin called Bt toxin. The Bt gene is
derived from a bacterium called Bacillus thuringiensis and produces a toxin that disrupts gut function in
the larvae (caterpillars) of certain insects that are crop pests. The gene that produces Bt toxin is introduced
into such plants by recombinant DNA technology, and results in the selective killing of crop-feeding
insects. This development has had a major economic impact and reduced the expenses of pesticides used
per year and has increased the longevity and success of several crops.

https://knowgenetics.org/recombinant-dna-technology/