Document1
Page 1 of 7
Molecular biology (and the use of E. coli as a model
organism) grew out of two fields of study:
1. A field that was concerned with the regulation of biochemical
processes (and later genetic processes) using (at first) the lac system of E.
coli.
•As a consequence of E. coli being an important organism from the
beginning of what is now molecular genetics, we know more about it than
we do about any other organism on the planet.
•Much of that information can be used as a foundation to study other
organisms because of the universality of biochemical and genetic
processes.
2. A field concerned with how bacterial viruses replicate (T-even and
lambda were main models). This field was started by physicists interested in
biological information propagation.
Why use bacteria (E. coli) to do genetics?
Some old reasons:
•The power of numbers: A billion cells can fit easily in a test tube the size
of the end of my finger (1 ml). A billion fruit flies would take a minimum
of 109 cm3 (10m3) which is the size of a fairly big lecture hall to hold (and
that doesn’t include food).
•They are single cells and haploid, that means that mutations affect the
whole organism and are easy to isolate.
•They grow fast: 1 cell to 1 billion cells in less than 12 hours.
•They are cheap to grow.
•They can grow on simple media with only a carbon source and a few salts.
• A large number of people world-wide also study E. coli.
•Extremely powerful genetics and molecular biology techniques
•Genome sequences of 2000 or so strains (not counting closely related
species like Salmonella, Shigella, Klebsiella See DOE_IMG site (sign in as
daniel.gage@uconn.edu Password is uconnmcb3617)
Document1
Page 2 of 7
E. coli Facts
•Can grow with a 20 minute doubling time. All components of every cell
are doubled in that time (with the exception of the chromosome).
•Strain MG1655 was the first E. coli sequenced and has 4,639,675 bp
encoding: 4132 proteins and at least 172 RNAs that don’t code proteins
•Other E. coli genomes are 4 to 6.2 million bp in size (Slide).
•About 25% of the genes have no known function
•Gram negative rod, about 1-3 um in length
•Colonies contain about 1 million cells each
A brief introduction to Genetics
What is genetics?
•Study of heredity and variation (old definition)
•Study of how biological information is retained, passed on, changed,
acquired, and regulated (newer definition).
Themes of bacterial genetics:
•How do cells grow
•Information processing:
Permanent: DNA, RNA
Temporary: signals, survival, pathogenicity
•Evolution
•Two ways to approach genetics:
>Forward (classic approach): Hunt for an interesting mutant trait
and then determine what caused it (look for phenotype, then investigate
genotype). Eg: find mutants resistant to cyanide--> then study the genes
responsible for the new (or broken) trait.
STRENGTH: Almost always find mutations that affect the trait you
want to study. Usually many are found.
WEAKNESS: It can sometimes be hard to figure out how the
mutations affect the trait. The gene > trait connection can be obscure or
complex
Document1
Page 3 of 7
>Reverse (came about after molecular biology): Find an
interesting gene, alter it and then see how the organism is changed
STRENGTH: The investigator has an idea about how the gene
functions before he/she disables it. So interpretation is often easy.
Example: Knockout stress protein gene expect cells to have trouble with
heat, metals, solvents, etc.
WEAKNESS: Very often altering the gene has no observable effect
on the organism. There are a number of reasons why this might happen.
Example: Knockout gene for a drug removal pump. Expect cells to be
drug sensitive. But if they are not—why not?  gene may be redundant,
protein may pump a drug other than the ones tested, pump may work or
be made only under special circumstances.
(Read: The Salvation of Doug and be prepared to discuss)
Some terms pertaining to growth/genetics
Complex medium: contains components of undefined chemical structure
(yeast extract, tryptone, etc). Slide with TY, LB
Defined medium: contains compounds of known chemical composition.
Sometimes called “minimal” medium when the number of ingredients is
fairly small. Slide with M9
Diploid-Two copies of each chromosome. Common in metazoans
Haploid-One copy of each chromosome. Common in bacteria
Wild type: A strain that contains no known mutations and was at some
point collected from the wild. Note that many “wild-type” strains have
been cultured under lab conditions so long that they have accumulated
mutations that allow them to do well under lab conditions (high growth
rates, good media, little competition). Most of these mutations are
unknown, and the strains are still referred to as wild-type. This also means
that my wild-type strain (eg Sinorhizobium meliloti Rm1021 may be different
from Rm1021 from another lab).
Document1
Page 4 of 7
Prototrophs: are organisms that can make everything they need from a
minimal salts medium-ie they can make all amino acids, nucleic acid
nucleotides, lipids, vitamins etc.
Auxotrophs: are organisms (often mutants) that cannot make everything
they need from a minimal salts medium-eg an E. coli trp mutant will need to
have tryptophan provided in the medium in order to grow.
Sugar requirements differ from auxotrophies and this can be confusing:
Lac– strains will not grow on media when lactose is provided as the sole
carbon source because the genes needed to transport or use lactose are
mutant.
So, a lac- trp- mutant can never grow on lactose (because it can’t use
it) and always needs tryptophan (because it can’t make it)
Genus+species are italicized and partially capitalized: Sinorhizobium meliloti
Genes are italicized and NOT capitalized: lacZ, trpB.
Proteins are NOT italicized and begin with a capital: LacZ, TrpB
This is important, and must be followed!! If you don’t follow you generate
confusion. For example, “The lab was provided with a tube containing E.
coli lacZ” is very different from “The lab was provided with a tube
containing E. coli LacZ.”
“A piece of TrpB was fused to trpA ” is either nonsense, or very interestng
chemistry, because it implies a protein fused to a piece of DNA
Phenotype/Genotype, nomenclature, etc
Genotype: is the way that a bacterial strain differs from a wild-type strain
of the same species. It is often written as a list of mutated genes.
Phenotype: The biological characteristics of a strain. Often refers to the
changes caused by an organism’s set of mutated genes.
Document1
Page 5 of 7
Slides of genotype/phenotype
Common types of mutations
Mutation: a heritable change in DNA sequence
Missense mutations: changes that result in an amino acid substitution.
These can cause a protein not to function, to function poorly, to function
the same, or sometimes to function better. The strength of the mutant
phenotype depends how different the new amino acid is from the original.
Example
-Missense mutations often have partial function--> that is they are
often “leaky”
Nonsense mutations: same as missense, but the DNA change results in a
stop codon, so protein synthesis terminates early. These usually are not
leaky, because the section of protein that is made is non-functional.
Deletion mutations: Mutations that remove a block of DNA, can be large
or small
Insertion mutation: DNA is inserted into a gene, can be large or small
Chemical mutagenesis: UV, MNNG, EMS discuss strengths/weak.
Tn mutagenesis. How to make a library. How to use it, how to find
the resulting mutations. Strengths/weak
Finding mutants
Two ways (one easy, the other hard) to find mutants:
1)Easy: Selection-the mutant of interest grows and all others die. Plate 109
cells on LB + Ampicillin. Only the few cells with mutations confering amp
resistance will grow.
Document1
Page 6 of 7
2)Harder: Screen-assume you want to find lac mutants. If you plate 109 cells
on M9 lactose + amino acids + vitamins, 999,999,000 will grow, and the
1000 or so lac mutants will not. So, that won’t work. How do you do it?
-plate ~500 colonies per plate on M9 glucose + amino acids+
vitamins and then test by replica plating onto M9 lactose. Most colonies
will grow, but every once and while one will not-->those are lac mutants.
Expect 1 per 106 cells screened if mutation rate is 10-9!! Is there a better
way?
Perhaps: Is there a toxic analog of lactose that will kill lac+ bacteria,
leaving lac- bateria alive? Yes: o-nitrophenyl-thiogalactoside. This kills
bacteria when it accumulates inside to high levels.
So, grow cells in a tube of M9+phenylgalactoside. Most wt cells will die
and then plate ~500 colonies on M9 + glucose +aa+vit., then replica plate
to M9 + lactose. You will find many more lac- cells now, because most wt
cells died. This is an enrichment.
Another famous example of an enrichment is penicillin enrichment-first
described by Lederberg and Lederberg (1952)
This is based on the fact that penicillin inhibits cell wall synthesis when
cells are growing and therefore causes cells to lyse when they are growing.
Non-growing cells are more resistant to penicillin attack. So, you can use
this technique to help find non-growing cells. This is very useful, for
example you could use this to find lys- cells that fail to grow in minimal
medium (you would also enrich for other auxotrophic mutants, such as ilemutants. How could you make sure you got mostly lys- mutants if that was
what you wanted? --> by including all a.a. except lysine--so ile- mutants
would grow, and be killed by penicillin, because isoleucine was provided..
3 Slides on penicillin enrichment based on Fitzgerald and Williams
Document1
Page 7 of 7
Complementation
Complementation is a technique where you fix a mutant
phenotype by providing cells with a fixed copy of the broken
(missing or mutant) gene. The fixed copy can be on a plasmid, in
the chromosome or carried on a bacteriophage.