Chapter 10: Bacterial Genetic Analysis and Manipulation

Pathogen

Early researchers in the field focused on microbes of practical

importance (organisms that causes disease)

auxotrophs

Allowed study of one gene based on its inability to synthesize a

particular nutrient - needs it in the growth media (since auxotrophs are unable to synthesize one or more organic compounds)

Replicon

Organization of bacterial genomes

Single chromosome and plasmid

wild type strain and mutant strain

wild type strains are naturally occurring, genetically normal form of a bacterium in a given population, representing the standard or reference genotype and phenotype whilst

mutant strains are are altered versions of the wild type, resulting from genetic changes (mutations) that can be spontaneous or induced, leading to different traits or behaviors

What is the goal?

To identify different alleles of genes (any one of two or more genes that may occur alternatively at a given site (locus) on a chromosome)

genotype

an organism describes its collection of alleles

of a given set of genes

gene

a three-letter abbreviation in italics, followed by a

capital letter to separate genes in the same pathway

protein

given the same three-letter designation but with the

first letter capitalized and no italics

phenotype

Changes in genes are often visible by changes in…. (which are physical appearance changes)

phenotypic selection

Use of a growth

medium that will

inhibit microbes lacking

the desired gene(s).

• Antibiotic selection is

commonly used

Phenotypic screening

Duplicate plates are

created. (B)

o The first is under full

nutritional support.

o The second lacks a

particular nutrient.

• Where a colony grows on

the fully supported plate,

but doesn’t grow on the

partial support plate, a

mutation has occurred

replica plating

Duplicate plates are created by the process of…

restriction enzymes

Allow cutting of specific DNA pieces (a required prerequisite

to moving or copying DNA fragments)

• Each RE recognizes a restriction site and cuts it.

o Often, the cuts are asymmetrical.

• Similar ends of cut DNA can be paired together.

• Paired ends can be tied, or ligated, by DNA ligase.

Cloning vectors

REs allow researchers to stitch together fragments of useful

DNA into recombinant molecules.

o Recombinant molecules can be used to “clone,” or make many

copies, of a bacterial gene of interest.

Plasmids

a small, circular DNA molecule that exists independently of chromosomal DNA and can replicate autonomously, often carrying genes that provide advantages such as antibiotic resistance.

plasmid cloning vectors

today, there are a number

of “common” plasmid

vectors with desirable traits

for easier gene cloning:

o Origin of replication

o Selectable marker gene

o Multiple cloning site(s)

o Small size

o High copy number

Recombination

Incorporation of

foreign DNA into the host cell’s

chromosome(s

Homologous recombination

Occurs when two identical (or

nearly identical) fragments of DNA

line up and exchange pieces.

Non-homologous recombination

ecombination of DNA pieces with little or no similarity

• Occurs in all forms of life

o Especially prevalent in viruses.

o Temperate bacteriophages undergo lysogeny by site-specific

recombination, inserting their genome at particular points in the

host cell DNA sequence.

Transformation

introduction of extracellular DNA directly into an organism

Conjugation

Transfer of DNA from cell to cell via direct

contact/sex pilus formation

Transduction

Sometimes during phage replication,

a virus will accidently package a

fragment of host cell DNA.

• When this “odd” virus infects the

next cell, it delivers that fragment

instead of viral DNA.

o The “odd” virus is usually unable to

replicate because it lacks the viral

genome.

• Homologous recombination must still

occur, though.