Download Developing a new genetic system in bacteria

Genetics in the real world:
Developing a new genetic system
in bacteria
Abigail Salyers
[email protected]
General advice
• Don’t start with methods development before you
have a specific biological question in mind!!!
– What are the most useful tools (transposon mutagenesis,
single crossover disruption, replicating plasmid, deletions,
fusions?)
– What to do first
– What can you do if you can’t get DNA into your strain?
• Powers of 10 are important!
• Be flexible.
• Be ready to optimize.
Essentials for a classical genetic
system
•
Means of isolating DNA and RNA!!
•
Means of introducing DNA into your organism
•
Transformation/electroporation
– Conjugation
•
Shuttle vector – replicates in E. coli and your organism
– Introduce cloned genes
– Complementation
– Increasing gene dosage (but naturally occurring plasmids are usually low
copy number – 1 - 10 copies per cell)
•
Suicide vector – replicates in E. coli but not in your organism
organism
– Single crossover or double crossover disruptions, deletions
– Transposon mutagenesis
Luxuries
• Reporter gene for constructing gene fusions
– Can do translational as well as transcriptional
fusions
– But, RT-PCR or microarray can be used to
measure gene expression directly; His-tagged
proteins can be detected in Western blots
(commercially available antibody)
• Transducing phage
– Useful for moving mutations into a fresh strain,
but can move mutations by insertion of genes
– May be difficult to find, especially if your
organism has a capsule
Genome sequence
• More realistic possibility today than ever before,
especially with 454 sequencing.
• Useful for
– Locating potentially important genes (by homology)
– Mapping genes you find by other methods (eg, cloning,
transposon mutatenesis) – find linked genes that may be
involved in your process
– Microarray possible
• Remember: all annotations are hypothetical and
need to be tested independently. Sequence is a
hypothesis generator, not proof!!!
Introducing DNA
• Shuttle vector – replicating plasmid with a
selectable marker
• Constructing a shuttle vector
– Many strains contain cryptic plasmids
– DNA sequencing to locate restriction sites, other
features (replication origin)
– Antibiotic resistance gene from the target
organism, if possible (functional promoter)
– Usually best to have a resistance gene with a
broad spectrum (tetM, ermB), minimal
background (eg, not a b-lactamase)
Introducing DNA (cont.)
• Suicide vector – does not replicate in your
organism
• Same features as shuttle vector, except lacks
replication origin that works in your
organism
– Transposon mutagenesis
– Single crossover disruptions
– Double crossover disruptions
Introducing DNA (cont.)
• Have a strong, clean selection – can detect 1 resistant cell in
108 susceptible ones
• Try electroporation first
– Buffer – whatever you freeze the strain in
– Optimize conditions to retain viability of recipient
– If arcing is a problem, try washing bacteria to remove ions trapped,
eg, in capsular material
• Conjugative transfer from E. coli (or some convenient donor)
– Has worked in cases where electroporation failed (probably due to
restriction enzymes)
– Plasmid vector needs a transfer origin (oriT)
– Mobilize with IncP plasmids (RK4, R751) – very promiscuous, high
frequency transfer
Introducing DNA (cont.)
• Need to optimize entry of DNA to get frequency of
plasmid entry as high as possible (preferably 10-3 per
donor or recipient)!!! Try different growth phases,
different ratios of DNA (transformation) or donor
(conjugation) to recipient
– Transposon mutagenesis frequency = frequency of transfer
X transposition frequency (10-3 or lower)
– Single crossover insertion by homologous recombination
(10-4 or lower)
Single crossover insertion for
gene disruption
• Clone internal segment of gene to be disrupted into
a suicide vector, introduce into organism and select
for marker on suicide plasmid
Cloned segment will be duplicated
May not be good for disrupting small genes – usually, need
> 200 bp to get detectable insertion frequency
• Disruption can have polar effect on downstream
genes
• Disruption can excise transiently, even in the
presence of a strong selection (10-4 or less) – “leaky
mutation”
Deletions
• Clone gene with deletion in it into a suicide vector
• Selection for marker on suicide vector most likely
lead to single crossover on one side or other of the
mutation
• Homologous recombination will either regenerate
the wild type (drat!) or the deletion (hooray!)
– Screeing for loss of marker on integrated plasmid – at least
104 colonies
– Selecting AGAINST marker on plasmid (eg., sucrase gene in
E. coli, other genes in other organisms)
Measuring transcription and
translation with fusions
•
Reporter gene – background activity has to be very low
•
Source of ribosome binding site
b-galactosidase activity very high in some bacteria
b-gluronidase (uidA) has worked in many bacteria (but be sure that gene
has good ribosome binding site)
– Fusing reporter gene with start codon (translational fusion) retains
ribosome binding site from target organism
– Fusing reporter gene with promoter usually replaces native ribosome
binding site with that of reporter gene (due to proximity of ribosome
binding site to start codon – need to add a good rbs for your organism
•
Detection method must work in your medium conditions
– X-gal, fluorescent green protein, luciferase do not work in most medium
used to grow anaerobes (reducing conditions)
– No plate screen
Constructing a transcriptional fusion
(Note that rbs is part of reporter gene)
Measuring transcription and translation –
bypassing fusions
• Transcription
– RT-PCR, RT-qPCR, microarrays
– Determination of operon structure by amplifying intergenic
regions
– Limitation is quality of RNA extracted from cells
• Translation
– Antibodies raised against theprotein of interest detect
protein on Western blot – expensive, time-consuming
– For tagged protein (e.g. His-tag), can buy antibody
– Limitation is whether tagged protein is functional in vivo (if
not, there is some question about stability, etc. of protein)