Topic 9: Microbial Genetics Flashcards
Reasons why bacteria are ideal genetic research tools
- Single chromosome and plasmids
- One chromosome for easy detection of mutations
- In early studies, nutritional mutants were used
- Allowed study of one gene based on its inability to use or produce a particular nutrient
Wild Type
- Strains of bacteria or archaea that are essentially same as when originally isolated from nature
- It is from wild type strains that mutants are generated for further analysis
Mutant
-Strain derived from wild type that contains mutation
- Named after phenotype, always in comparison to phenotype of original environmental strain
- Have genetic change that disrupts or alter the function that was associated with the wild type
Alleles
- Refer to gene variant associated with the mutant strain
- Can be gain of function, where a mutant can do a function the wild one couldn’t (ex: antibiotic resistant mutant vs antibiotic-sensitive wild type)
- Or, can be loss of function alleles (ex: antibiotic resistant wild type, antibiotic sensitive mutant)
- Change of function alleles (ex: yellow colonies for wild type and red colonies for mutants)
Auxotrophs
- Specific type of strain (either wild or mutant) that can’t make an organic compound that is required for growth
- Most common example of auxotrophic mutants are those that can’t make specific amino acids
- Also include strains that can’t make individual nucleotides of vitamins
Prototroph
- Related strain that is capable of producing the organic compound that auxotroph cant
- Usually parental strain that gave rise to the auxotrophic mutant
Genotype
- description of alleles within an organism, generally reflects differences from wild-type
Phenotype
- observable properties of a strain, ignores what genes are involved
Selection and Screening
Screening is more tedious than selection.
Phenotypic Selection:
- Used for selectable mutations (i.e., mutants grow under conditions that would kill wild-type cells)
- Useful in genetic research (e.g., to find antibiotic-resistant strains)
Screening:
- Used for nonselectable mutations (i.e., mutants that do not have a growth advantage over wild-type cells)
- Large numbers of colonies must be screened (tedious)
Screening by comparing plates:
- Duplicate plates are created and one lacks a particular nutrient
- a mutation has occurred where a colony grows on the full support plate but doesn’t on the partial support plate
Screening on a single plate:
- Use differential media to look for a difference in colour
Replica Plating and Patching
- Increase the throughput of colonies screened for mutants of interest by “copying” cells from a master plate onto differential media with either a wooden block (replica plating) or by robotic or manual colony transfers (patching, colonies on gridded plate, compare two)
Restriction and Modification Enzymes
- Restriction enzymes cut DNA at a specific recognition site.
- Recognition sites are usually palindromic
- Similar ends of cut DNA can be paired together and ligated
- Restriction enzymes are always paired with modification enzymes
- Often in a single operon
- Recognize the same site as the paired restriction enzyme
- Methyltransferase activity protects DNA from restriction enzyme activity.
Explain how plasmids can be used to clone DNA for screening or selection
- Restriction enzymes allow researchers to stitch together fragments of useful DNA into recombinant molecules
– Recombinant molecules can be used to clone a bacterial gene of interest - Vectors are used to insert a recombinant DNA molecule into a recipient host bacterial cell:
– Plasmid vectors
– Phage vectors
– Cosmids
Plasmid Cloning Vectors
- Plasmid Cloning Vectors were first used in the 1970s by Cohen at Stanford.
- He cut fragments from two plasmids carrying antibiotic resistance genes with the same restriction enzyme
- The transformed strain exhibited traits from both plasmids
- Today plasmid cloning vectors are engineered with desirable plasmid traits for easier gene cloning:
— Origin of replication
— Selectable marker gene
— Multiple cloning site
— Small size
— High copy number
Alternate hosts
- Certain host cell types may be restricted by the origin of replication
- Shuttle-vector plasmids have multiple types of origins, which expands the range of host cell types the plasmids can be inserted into.
Phage Vectors
- Mix viral DNA with fragment of interest
- Lysogenic lambda phage can carry ~20-kb fragments
Cosmids
- Phage genomes that omit nearly all the phage DNA, leaving more room for the fragment
- critical phage cos packaging recognition sites remain
- Other elements include a multiple cloning site and an antibiotic selection marker
- Cosmids can typically carry 35‒45 kb fragments
Transformation
- Introduction of extracellular DNA directly into an organism
- Doesn’t require cell-to-cell contact
- Some bacteria are naturally competent for transformation, take directly from environment
- Other bacteria can be artificially induced to become competent by:
— Treatment with calcium cations
— Electroporation
Conjugation
Conjugation: the transfer of DNA from cell to cell via direct contact/sex pilus formation.
Mechanism of Conjugation
– F plasmid carries the gene to form sex pilus “bridge” between two cells
– F plasmid can be copied and sent across the bridge (origin of transfer first) into a recipient cell
– Turns an F– cell into an F+ cell
Hfr Strains
– The F plasmid integrates into the host chromosome by homologous recombination, thus it is an episome (DNA that can integrate into the chromosome or exist autonomously).
— Creates new opportunities to pass genetic information
– Incorporated F plasmid sends the host cell DNA next to its incorporation site across the sex pilus.
— Can be used to “map” the location of genes in the host chromosome
— Generation of F′ plasmids
Triparental Conjugation
– Conjugation can still occur using recombinant plasmid lacking the required tra gene and a helper plasmid with the gene.
Transduction
- Virus accidently packages a fragment of host cell DNA
- Virus delivers random host genome fragment instead of viral DNA to the next cell
- Virus is usually unable to replicate because it lacks the viral genome
- Few transducing phage when they occur
- Homologous recombination must occur to integrate into a recipient genome
Specialized Transduction
- Lysogenic bacteriophage integrates at specific site
- Virus excises incorrectly, including some host genome
- All virus particles are transducing phage following lysis
- Homologous recombination must occur to integrate into a recipient genome
explain the following conjugation terminology: F+, F−, F′, Hfr, tra, oriT
F+: F-plasmid found autonomously within the cell
F-: cell that does not contain a F-plasmid
F′: an incorporated F plasmid excises itself by homologous recombination
Hfr: high frequency of recombination
- F-plasmid is found incorporated into the chromosome
- can then transfer the entire chromosome over the mating bridge during conjugation (from a donor to a recipient)
- Can have multiple insertion sites dependant on homology
- Can produce F’ bacteria, which can conjugate
- Can be used to map the location of genes in the host chromosome
Tra: encode all of the proteins needed for conjugation
oriT: origin of transfer, directional
explain how mobile genetic elements can mobilize DNA through replicative and non-replicative transposition
Replicative transposition:
- Copies the element and moves the copy to another location
- Has res site and resolvase gene
Non-replicative transposition:
- Cuts and pastes the element into a new location
Application of Transduction
- Historically, co-transduction frequency was used to map bacterial genomes; genes that were closer to a known “marker” gene would be transduced with that marker more frequently than ones farther away
- Can also be used to modify bacteria
