ALTERATION

OF CELLULAR INFORMATION

EVOLVING DESIRABLE BIOCHEMICAL ACTIVITIES THROUGH MUTATIUON AND SELECTION

• Although the cell has a well-developed system to prevent errors in DNA replication and an active
repair system to correct damage to a DNA molecule, mistakes can still occur. These mistakes are
called mutations.

• The genotype is the set of genes in our DNA which is responsible for a particular trait.
Genotypes can only be determined by biological tests, not observations. Genotype is an
inherited trait and hereditary information passed by the parents determines genotype

• The phenotype is the physical expression, or characteristics, of that trait. Phenotype is what
you see the visible or observable expression of the results of genes, combined with the
environmental influence on an organism's appearance or behavior.

• A mutation is a genotypic change and is irreversible.

How Mutations Occur

• Most mutations occur because of mistakes in DNA synthesis. One common form is a point
mutation.

• Point mutation or substitution is a genetic mutation where a single nucleotide base is changed,
inserted or deleted from a sequence of DNA or RNA. Point mutations have a variety of effects on
the downstream protein product consequences that are moderately predictable based upon the
specifics of the mutation.

• SILENT MUTATION- are mutations in DNA that do not have an observable effect on the
organism's phenotype.

• NONSENSE MUTATION-is a point mutation in a sequence of DNA that results in a

premature stop codon, or a point-nonsense codon in the transcribed mRNA, and in a
truncated, incomplete, and usually nonfunctional protein product..

 • Deletion Mutation (also called gene deletion, deficiency, or deletion mutation) is a mutation (a
genetic aberration) in which a part of a chromosome or a sequence of DNA is lost during DNA
replication. Any number of nucleotides can be deleted, from a single base to an entire piece of
chromosome. By deleting or adding one or more bases, we can alter the whole composition of a
protein, not just a single amino acid. A deletion can shift the reading frame when translating the
resulting mRNA.

• Frameshift mutations arise when the normal sequence of codons (sequence of three
nucleotides which together form a unit of genetic code) is disrupted by the insertion or
deletion of one or more nucleotides, provided that the number of nucleotides added or
removed is not a multiple of three. This can result in the incorporation of many incorrect
amino acids into the protein.

• Back mutations or reversions are possible. Revertants are cells for which the original wild-type
phenotype has been restored. Restoration of a function can occur due to a direct change at the
original mutation. If the original mutation was CAA to UAA, then a second mutation for UAA to
CAA restores the original genotype and phenotype by the means of a suppressor mutation.

Selecting for Desirable Mutants

• Mutants can serve as powerful tools to better understand cell physiology; they are also valuable
as industrial organisms because mutation can be used to alter metabolic regulation and cause
overproduction of a desired compound. Methods to induce mutations and then select for
mutants are important tools for catalyst development in bioprocessing.

• Natural (spontaneous) rates of mutation vary greatly from gene to gene.

• Chemical agents (mutagens) and/or radiation are often used in the laboratory to increase
mutation rates. Mutagens are nonspecific and may affect any gene.

• CLASSIFICATION OF MUTATION

• Selectable mutation confers upon the mutant an advantage for growth or survival
under a specific set of environmental conditions; thus, the mutant can grow and the
wild type will die.
 • Unselectable mutant requires a cell-by-cell examination to find a mutant with the
desired characteristics

• DIRECT SELECTION

• Direct selection involves inoculating cells onto a medium on which the mutant, but not
the parent, can grow. For example, mutants resistant to the antibiotic streptomycin can
be easily selected directly by inoculating cells onto a medium containing streptomycin.
Only the rare resistant cells in the population will form a colony. Antimicrobial-resistant
mutants are usually very easy to isolate by direct selection.

• INDIRECT SELECTION

• Indirect selection is required to isolate an auxotrophic mutant, one that requires a

growth factor, such as histidine, which the parent strain does not. On a medium that
contains histidine, both the parent and the histidine auxotroph grow. On a medium
lacking histidine, the histidine-requiring mutant will not grow, but the parent will. There
is no medium on which the mutant will grow and the parent will not.

• Replica plating is a microbiological technique in which one or more secondary Petri

plates containing different solid (agar-based) selective growth media (lacking nutrients
or containing chemical growth inhibitors such as antibiotics) are inoculated with the
same colonies of microorganisms from a primary plate (or master dish), reproducing the
original spatial pattern of colonies. The purpose of replica plating is to be able to
compare the master plate and any secondary plates, typically to screen for a desired
phenotype. Common screenable phenotypes include auxotrophy and antibiotic
resistance.

Gene transfer is defined as the introduction of genetic material into a living cell in order to
induce and achieve synthesis of a desired gene product. It occurs by the conveyance of a DNA
from a donor cell to a recipient cell.

Two Major Categories:

• Horizontal Gene Transfer

► Transfer of genetic material between unrelated individuals

• Vertical Gene Transfer
► Transfer of genetic material from parental organism to offspring

Natural Mechanisms
• Horizontal Gene Transfer
►Transformation - Transformation is a form of genetic recombination in which a DNA
 fragment from a dead, degraded bacterium enters a competent recipient bacterium and
is exchanged for a piece of DNA of the recipient. Typically, this involves similar bacterial
strains or strains of the same bacterial species.

► Transduction - a process of genetic recombination in bacteria in which genes from a
host cell (bacterium) are incorporated into the genome of a bacteriophage and then
carried to another host cell when the bacteriophage initiates another cycle of infection.

► Bacterial Conjugation - the transfer of genetic material between bacterial cells by direct cell-
to-cell contact or by a bridge-like connection between two cells.
 • Vertical Gene Transfer
► Reproduction

GENETIC ENGINEERING

It is a set of tools and not a scientific discipline. Although difficult to define precisely, it involves the
manipulation of DNA outside the cell to create artificial genes or novel combinations of genes with
predesigned control elements.

Basic Elements of Genetic Engineering

Recombinant DNA techniques

the ability to isolate genes from one organism and recombine the isolated gene with other DNA that can
be propagated in a similar or unrelated host. Most of our discussion is drawn from approaches for
genetically engineering bacteria.

Obtaining the gene of interest

A simple, brute-force approach is shotgun cloning. Here the DNA from the donor organism is cut into
fragments using restriction enzymes.

Most often, such a shotgun approach is very inefficient. More specific approaches use hybridization.
A probe can be synthesized chemically to be complementary to a portion of the gene. The probe is
usually much shorter than the gene but sufficiently long that it is unlikely for other genes to have the
same complementary DNA sequence.

Large amounts of a target gene can be generated by a technique called the polymerase chain
reaction(PCR). Large amounts of a target gene can be generated by a technique called the polymerase
chain reaction(PCR).

Inserting the gene into DNA

Once the desired gene is isolated or made, it can be inserted into a small piece of carrier DNA called
a vector. Typically, vectors are plasmids, although temperate viruses can be used. The process for
preparing the donor DNA and vector for recombination and the actual joining of the DNA segments
usually requires special enzymes

Genome Engineering

- It is also known as GENOME EDITING.

- refers to the collection of strategies and techniques developed in recent years for the targeted,
specific modification of the genetic information—or genome—of living organisms.

Multiplex automated genome engineering (MAGE) is a method for introducing targeted

modifications directly into the chromosome of E. coli using synthetic single-stranded DNA (ssDNA).
 The process is cyclical and begins with transformation of ssDNA by electroporation followed by
outgrowth, during which bacteriophage homologous recombination enzymes mediate annealing of
ssDNAs to their genomic targets.

Genomics

• the study of whole genomes of organisms, and incorporates elements from genetics.

Experimental Techniques

• Genomics uses a combination of recombinant DNA, DNA sequencing methods, and

bioinformatics to sequence, assemble, and analyse the structure and function of genomes.

• Historically, the nucleotide sequence of DNA fragments was determined on a sequencing gel by
a process called Sanger sequencing, named after its inventor Frederick Sanger.

• Fred Sanger's group established techniques of sequencing, genome mapping, data storage, and
bioinformatics analyses in the 1970s and 1980s. This work paved the way for the human
genome project in the 1990s

Other technologies that make large-scale whole-genome sequencing accessible and practical

• Nanopores

• Microarrays

• Transcriptomics