Bacterial genetics III

Engineering genes and cells

PMB/MCB 112

Because Bio 1A is a prerequisite for this course,

Im going to assume that you are familiar with the
following techniques:
using PCR to amplify a gene of interest
using restriction enzymes to cut DNA at specific sites
using ligase to covalently link two pieces of DNA
with compatible ends
inserting a gene of interest into a plasmid with a selectable
marker
If you are NOT familiar with these topics, please consult
Brock!

Why would we want to express a particular gene from

a plasmid in a bacterial host?
1) Move the gene into a different species or a different
mutant background
2) Express a different allele of the gene than the WT allele
3) Complement a genomic mutation with the WT allele
of the gene on a plasmid
4) Change the regulation of the gene (turn it off/on,
increase its expression level)
All of these techniques are used to understand
the function of the gene/protein in question

Complementation of mutant phenotypes using genes carried

on plasmids
In any mutant made using UV or ,
a chemical mutagen,
x
how do I find the affected gene?
Transform mutant with genomic DNA library from WT parent
1) Select for presence of the plasmid
2) Select or screen for colonies that have
the wild-type phenotype restored
Any colonies with the WT phenotype
contain a plasmid expressing the WT
gene corresponding to the mutated
site in the chromosome

Changing gene regulation with an inducible promoter

Some proteins are needed in the cell only under specific
conditions. These proteins are typically not expressed
all the time.

Pxyl

xylX

xylA

xylB

xylC

Example: when Caulobacter senses the sugar xylose,

it turns on genes (xylXABC) that allow it to use xylose
as a carbon source
The xylose-inducible promoter (Pxyl) is only active in the
presence of xylose.

Using molecular biology techniques, we can place Pxyl

in front of any gene of interest (yfg).

Pxyl

xylX

Pxyl

xylA

xylB

xylC

yfg

If we put this gene back into Caulobacter (on a plasmid),

then we will be able to turn its expression on and off
by adding or removing xylose, and we will be able to
examine the consequences for the cell.

RANDOM mutagenesis
When do I use it?
when I dont know AT ALL what genes are involved in
my process of interest, or when I have no sequence
information about my organism
Tools: chemical mutagens, UV radiation, transposons
TARGETED mutagenesis:
When do I use it?
when I can predict what gene(s) might be involved
in my process of interest (I have candidate genes to test)
Tools: you MUST have sequence information about the
genes you are targeting, or a whole genome sequence
of your organism

Creating knockout mutations by targeted gene disruption

orfA
plasmid

lines represent various restriction enzyme sites

genomic region
surrounding orfA 1) PCR orfA and surrounding region and clone it into a plasmid

2) Cut orfA with restriction enzymes

3) Replace internal region with antibiotic resistance gene

KanR

Leave 500-1000 bp of chromosomal sequence at each end so that

homologous recombination can occur between these pieces of DNA
and identical sequences in the chromosome

Transform competent
bacteria with KO plasmid
To ensure recombination,
you can linearize the plasmid
before transformation by
cutting with a restriction enzyme
(BamHI in this example)
Or you can use a plasmid that
doesnt replicate in the host
where you are making the KO
The only way a cell could
become KanR is by
recombination of the disrupted
gene into the chromosome,
replacing the wild-type copy

When doesnt this work?

When the gene you are trying to disrupt is ESSENTIAL.
How do you learn the null phenotype of an essential gene?
1) Create a plasmid with the essential gene driven by
an inducible promoter
2) Transform this plasmid into your host strain and keep the
gene ON using the appropriate inducer
3) With the plasmid gene ON, knock out the chromosomal
copy of the gene using targeted gene disruption
4) When the plasmid gene is the only copy, turn it OFF by
removing the inducer
5) Wait for the protein to be go away and observe the cells
to see what happens in the absence of the
essential gene/protein

Forward genetics

Reverse genetics

Make random mutations

Make directed mutations

in genes of choice

Must select or screen

through105-109 mutants
to find ones with the
desired phenotype

Must test each mutant in

the set of 10-1000 mutants
for the desired phenotype

Transposon mutagenesis is RANDOM

You cant say Ill mutate gene X specifically using a transposon.
You would have to mutagenize at RANDOM with a transposon,
then 1) select mutants that had received a transposon and
2) select or screen for mutants with your desired PHENOTYPE.
This does NOT guarantee a mutation in a specific gene.
Transposons have inverted repeats at their ends, and these are
required for the transposase enzyme to catalyze insertion into the
chromosome.
Transposons DO NOT insert at places where there are already
inverted repeats!!! They can insert into ANY DNA sequence.
Transposons DO NOT insert into target DNA by homologous
recombination. No homology is required between the transposon
and the target, and RecA is not used. The transposase catalyzes
the reaction.

To make a targeted gene knockout, you have to know

the sequence of the gene you are disrupting
This is NOT a random technique. You know the gene you are
knocking out, but you dont know what phenotype you will get.
PCR the gene out of the chromosome and clone into a plasmid.
Use restriction enzymes to cut out the center (or all) of the open
reading frame and replace it with an antibiotic resistance
gene.
Leave 500-1000 bp of chromosome sequence on each side
for homologous recombination.
Make sure plasmid cant replicate in the host.
The knockout construct is integrated into the genome by
homologous recombination (using RecA), with the antibiotic
resistance gene replacing the normal gene.