Lecture 2: Methods in Bacteriology Flashcards
Insertional inactivation (suicide plasmids)
- suicide plasmids contain an ORI (origin of replication, the place where DNA starts replicating) that is NOT compatible with host cell machinery
- this means they are unable to replicate within the target organism
- it will be lost from the cell (as the population grows and divides) unless it integrates into the chromosome
- to create: identify target gene, create a PCR primer that matches a section of the gene, PCR amplify it, ligate that gene and place it into a plasmid (suicide plasmid) - ususally in Ecoli, with an antibiotic resistance gene
Making suicide plasmids
- to propagate suicide plasmids in the lab, specialist Ecoli cells are used that carry the correct proteins that recognise the ORI on the suicide plasmid
- this allows the plasmid to replicate within a population of Ecoli cells
- the suicide plasmid is introduced to the Ecoli cell through TRANSFORMATION (Ecoli is treated to be more permeable to DNA)
Investigating bacterial gene function
- inactivating and deleting genes is how their function is discovered
- Creation of a mutant (through inact./deletion)
- Assess mutant for phenotypic variance
- Restore gene function through complementation
- homologous recombination underlies mutagenesis strategies
Gene complementation tests
- complementation occurs when two strains of an organism with different homozygous recessive mutations that produce the same mutant phenotype (for example, a change in wing structure in flies) produce offspring with the wild-type phenotype when mated or crossed
Suicide plasmids can be transferred by two methods
- Conjugation
Donor and recipient strains are mixed together to allow conjugation to occur - Electroporation
Plasmid DNA is extracted and mixed directly with our recipient
A brief electrical pulse is applied to the mixture, which increases the permeability of the bacterial cell and promotes plasmid uptake
When a suicide plasmid is introduced to the recipient cell the cell is called a
Transconjugant (transient, cannot be maintained in the cell)
- However, the plasmid cannot replicate; it must either integrate into the chromosome, or be lost from the cell
- It integrates through homologous recombination
Integration of plasmid into the chromosome
- Plasmid integrates into target gene through homologous recombination
- Disrupts the gene, and thus results in loss of the gene product
- plasmid uncircles and is added to the DNA, the gene is split in two and the plasmid DNA lies in between the two halves - disrupts the original DNA as well, inactivating it
- Resulting transconjugant can be selected for on the basis of the antibiotic resistance marker carried in the plasmid
Pros and cons of insertional inactivation
Pros:
Quick and simple, as reliant on a single homologous recombination step
Cons:
Plasmid is retained within the target bacterium, thus conferring antibiotic resistance; limits subsequent genetic manipulation
Insertional inactivation may have “polar effects” on downstream genes
Polar effects of insertional inactivation
- Prokaryotic genes frequently occur in operons, with a single RNA transcript containing multiple genes and thus encoding multiple proteins
- Insertional inactivation of one gene within an operon may affect function of downstream genes within the same operon – “polar effects”
- you don’t know whether the phenotype is from the gene you disrupted or from polar effects (you can test this by adding back in the functioning gene, to see whether it restores phenotype)
- Creation of unmarked deletion mutants is preferred
Insertional Inactivation vs Unmarked Deletion
Insertional inactivation:
For insertional inactivation, the fragment cloned into the suicide vector is internal to the target gene
Unmarked deletion:
To create unmarked gene deletions, we clone fragments that flank the target gene (i.e. regions upstream and downstream of the gene) (PCR primers for these)
Suicide vector also contains a “counterselectable marker”
Creating unmarked deletion plasmids
- The suicide plasmid contains both upstream and downstream regions, integrate through conjugation or electroporation
- The first recombination event can take place with either region (but not both, eg. two upstream regions)
- This “first cross-over” results in the integration of the resistance gene (e.g. Tp) and the sacB counterselectable marker into the chromosome
- The sacB counterselectable marker encodes a levansucrase enzyme
- Converts sucrose to a toxic product that kills the bacterial cell
- Plating the first cross-overs on sucrose selects against the presence of the sacB gene (i.e. counter-selects)
- This promotes a second homologous recombination event to “throw out” the sacB-containing plasmid
- recombination between homologous regions throws out sacB: eliminates the plasmid DNA (revert to WT) OR downstream cross over results in UNMARKED DELETION (also does not carry AB resistance genes)
Pros and cons of unmarked deletions
Pros:
Should be free of polar effects on downstream genes
The resulting mutant does not carry any antibiotic resistance markers
Multiple genes can be deleted within the same strain (by sequential deletion of different target genes) because no accumulation of AB resistance genes
Cons:
More time-consuming, challenging
Counter-selection can prove problematic
Importance of complementation
- A critical step, irrespective of whether mutant was generated by insertional inactivation or gene deletion
- The entire sequence of the target gene is cloned into an expression plasmid that is introduced into the mutant strain
- By re-introducing a functional copy of the gene, we should restore any phenotypes back to the “wildtype”, thus verifying the role of that gene
Various factors can impact on success of complementation:
- Plasmid copy number (high or low)
- Expression level from the plasmid (native or non-native promoter?)
- Inducible or constitutive expression?
Identifying essential genes
There is great interest in identifying genes that are essential for bacterial growth and survival as potential antimicrobial targets
Neither insertional inactivation or unmarked deletion methods for mutagenesis can identify such essential genes
Why not?
- Can’t inactivate or delete a gene that is essential (it won’t grow)
- make conditional mutants instead
Identifying essential genes by conditional mutagenesis
For insertional inactivation, the fragment cloned into the suicide vector is internal to the target gene
- To create a conditional mutant, we clone a fragment corresponding to the 5’ end of the gene, starting precisely at the start codon
- This fragment is cloned into a suicide plasmid harbouring an inducible promoter immediately upstream of the fragment (e.g. rhamnose (sugar) promotor)
- Target gene is now immediately adjacent to and under the control of a rhamnose-inducible promoter
