Genetics in the real world: Developing a new genetic system in bacteria Abigail Salyers

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 – 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  -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 10 8 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 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 10 4 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  -galactosidase activity very high in some bacteria  -gluronidase (uidA) has worked in many bacteria (but be sure that gene has good ribosome binding site) Source of 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)