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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)