L3 – Bacterial Recombineering, Virulence Factor Identification and Characterisation Flashcards
What is recombineering?
Recombinering is a genetic engineering technique that utilises homologous recombination, often mediated by bacteriophage lambda proteins, to modify DNA without relying on restriction enzymes and ligases.
How does recombineering differ from traditional recombinant DNA technology?
It bypasses the need for restriction sites and in vitro ligation by exploiting the cell’s natural homologous recombination mechanisms.
Why is short DNA homology important in recombineering?
It directs the recombination event by providing regions of similarity that the recombination proteins can recognise.
What natural process is recombineering based on?
It is based on the natural repair mechanism of homologous recombination.
What is the primary aim of a gene knockout?
To delete or disrupt a functional gene while leaving surrounding genomic regions intact.
How is a common knockout achieved using recombineering?
By replacing the target gene with an antibiotic resistance cassette flanked by regions of homology.
What are key steps in constructing a knockout cassette?
PCR amplification of upstream and downstream flanking regions, excision of the resistance cassette from a donor plasmid, and ligation into a cloning vector.
What methods are used to confirm a successful knockout?
PCR screening and protein detection methods such as Western blot or ELISA.
What is a knock in strategy in the context of bacterial genetics?
It involves reintroducing a gene, often into a different genomic location or on a plasmid, to restore or study its function.
Why might knock in be necessary in virulence studies?
It is used to complement a knockout or to study essential genes whose loss would otherwise be lethal.
What considerations must be taken into account when performing a knock in?
Epigenetic effects, post-translational modifications, codon bias, and matching expression levels to wild-type conditions.
How does knock in help in verifying the specificity of knockout phenotypes?
It serves as a control to ensure that any observed phenotype is due solely to the gene deletion.
What are critical factors in selecting a recombinant protein expression system?
Choice of cloning method, promoter selection, tag type and location, and the suitability of the host (prokaryotic vs. eukaryotic).
How is cDNA amplification relevant to recombinant protein production?
cDNA amplification ensures intron-free templates, which is vital for prokaryotic expression systems.
What is the purpose of using fusion tags in protein expression?
Fusion tags facilitate protein purification and may improve solubility.
In the context of bacterial virulence, how can recombineering help elucidate bacteria–host interactions?
It allows for the precise modification of virulence factors, enabling the study of their roles in receptor binding and immune evasion.
How do bacteriophage lambda proteins (e.g. Redα, Redβ, Gam) facilitate recombineering?
They mediate homologous recombination by processing and annealing short homology regions, enabling efficient genomic modifications
What advantages does recombineering offer over traditional cloning methods?
It permits seamless modifications without dependence on restriction sites and enables direct manipulation of chromosomal DNA.
How does recombineering enhance functional studies of bacterial virulence factors?
Its precision and high efficiency allow for targeted mutations, enabling detailed analysis of individual gene functions.
What are potential pitfalls when performing recombineering?
Off-target recombination, unintended polar effects on neighbouring genes, and instability of the engineered construct are key concerns.
How can recombineering be combined with site-directed mutagenesis?
By using oligonucleotides with specific mutations in short homology arms, one can precisely alter amino acids in target proteins.
What is the role of selection markers in recombineering experiments?
They help isolate successfully modified clones by conferring antibiotic resistance or other selectable traits.
How can conditional knockouts be generated using recombineering?
By inserting inducible cassettes that allow gene expression to be toggled on or off under defined conditions.
Why are short homology regions (≈50 bp) sufficient for recombineering?
They provide just enough sequence identity for the recombination machinery to align and exchange DNA segments without the complexity of long homologies.
What are the key benefits of recombineering over traditional genetic engineering methods?
It eliminates the need for restriction enzymes and ligation, allowing for direct genetic modifications using homologous sequences.
Which recombinase is commonly used in recombineering?
Bacteriophage lambda Red recombinase.
How does recombineering allow for seamless genetic modifications?
By using short homology arms, recombineering enables precise modifications without introducing unwanted sequences.
Why is homologous recombination crucial in recombineering?
It ensures that the inserted DNA integrates correctly into the bacterial genome.
What is the role of exonucleases in recombineering?
They degrade DNA ends to create single-stranded overhangs, allowing for strand invasion and recombination.
How does recombineering facilitate gene knockouts in bacteria?
It replaces the target gene with an antibiotic resistance cassette through homologous recombination.
What is an antibiotic resistance cassette, and why is it used in recombineering?
A selectable marker that allows researchers to identify bacteria that have successfully undergone genetic modification.
Why is plasmid linearization important before transformation in recombineering experiments?
It ensures that the recombined DNA is correctly processed and integrated into the bacterial genome.
What is complementation, and why is it used in bacterial genetics?
It involves reintroducing a functional copy of a gene to confirm that observed phenotypic changes are due to its deletion.
How does recombineering support the study of bacterial virulence factors?
It enables the targeted deletion or modification of genes involved in bacterial virulence and pathogenesis.
Which bacterial species are commonly used in recombineering-based pathogenesis studies?
Neisseria meningitidis, Haemophilus influenzae, and Moraxella catarrhalis.
How can recombineering contribute to vaccine development?
By modifying bacterial surface proteins, researchers can study immune interactions and develop potential vaccine targets.
What are adhesins, and how can recombineering be used to study them?
Surface proteins that help bacteria attach to host cells; recombineering can generate mutants to study their function.
What role do CEACAM receptors play in bacterial pathogenesis?
They serve as binding sites for pathogenic bacteria, facilitating adhesion and infection.
How does recombineering help in studying bacterial interactions with immune cells?
By generating bacterial mutants lacking specific immune-interacting proteins, researchers can analyze host-pathogen interactions.
What are some challenges faced when using recombineering in bacterial genetics?
Off-target recombination, low recombination efficiency, and unintended mutations affecting nearby genes.
How does recombineering enable controlled gene expression in bacteria?
By inserting regulatory elements, researchers can control when and how a gene is expressed.
What strategies can be used to validate the effects of genetic modifications made through recombineering?
PCR, Western blotting, transcriptomics, and functional assays to confirm gene expression and phenotypic effects.
How can recombineering be applied to study protein function?
It enables the tagging, overexpression, or deletion of genes to study their role in bacterial metabolism and pathogenesis.
What are potential future applications of recombineering in microbiology research?
Potential applications include synthetic biology, antibiotic resistance studies, and engineering bacteria for therapeutic use.
