L3 – Bacterial Recombineering, Virulence Factor Identification and Characterisation

Question 1

What natural process is recombineering based on?

Answer 1

It is based on the natural repair mechanism of homologous recombination.

Question 1

What are key steps in constructing a knockout cassette?

Answer 1

PCR amplification of upstream and downstream flanking regions, excision of the resistance cassette from a donor plasmid, and ligation into a cloning vector.

Question 2

What is a knock in strategy in the context of bacterial genetics?

Answer 2

It involves reintroducing a gene, often into a different genomic location or on a plasmid, to restore or study its function.

Question 3

Why might knock in be necessary in virulence studies?

Answer 3

It is used to complement a knockout or to study essential genes whose loss would otherwise be lethal.

Question 4

What considerations must be taken into account when performing a knock in?

Answer 4

Epigenetic effects, post-translational modifications, codon bias, and matching expression levels to wild-type conditions.

Question 5

What are critical factors in selecting a recombinant protein expression system?

Answer 5

Choice of cloning method, promoter selection, tag type and location, and the suitability of the host (prokaryotic vs. eukaryotic).

Question 6

How is cDNA amplification relevant to recombinant protein production?

Answer 6

cDNA amplification ensures intron-free templates, which is vital for prokaryotic expression systems.

Question 7

What is the purpose of using fusion tags in protein expression?

Answer 7

Fusion tags facilitate protein purification and may improve solubility.

Question 8

How do bacteriophage lambda proteins (e.g. Redα, Redβ, Gam) facilitate recombineering?

Answer 8

They mediate homologous recombination by processing and annealing short homology regions, enabling efficient genomic modifications

Question 9

How does recombineering differ from traditional genetic engineering, and what are its key advantages?

Answer 9

It eliminates the need for restriction enzymes and ligation, allowing for direct genetic modifications using homologous sequences.

Question 10

What are potential pitfalls when performing recombineering?

Answer 10

Off-target recombination, unintended polar effects on neighbouring genes, and instability of the engineered construct are key concerns.

Question 11

How can recombineering be combined with site-directed mutagenesis?

Answer 11

By using oligonucleotides with specific mutations in short homology arms, one can precisely alter amino acids in target proteins.

Question 12

What is the role of selection markers in recombineering experiments?

Answer 12

They help isolate successfully modified clones by conferring antibiotic resistance or other selectable traits.

Question 13

How can conditional knockouts be generated using recombineering?

Answer 13

By inserting inducible cassettes that allow gene expression to be toggled on or off under defined conditions.

Question 14

Why are short homology regions (≈50 bp) sufficient for recombineering?

Answer 14

They provide just enough sequence identity for the recombination machinery to align and exchange DNA segments without the complexity of long homologies.

Question 15

Which recombinase is commonly used in recombineering?

Answer 15

Bacteriophage lambda Red recombinase.

Question 16

How does recombineering allow for seamless genetic modifications?

Answer 16

By using short homology arms, recombineering enables precise modifications without introducing unwanted sequences.

Question 17

Why is homologous recombination crucial in recombineering?

Answer 17

It ensures that the inserted DNA integrates correctly into the bacterial genome.

Question 18

What is the role of exonucleases in recombineering?

Answer 18

They degrade DNA ends to create single-stranded overhangs, allowing for strand invasion and recombination.

Question 19

How does recombineering facilitate gene knockouts in bacteria?

Answer 19

It replaces the target gene with an antibiotic resistance cassette through homologous recombination.

Question 20

What is an antibiotic resistance cassette, and why is it used in recombineering?

Answer 20

A selectable marker that allows researchers to identify bacteria that have successfully undergone genetic modification.

Question 21

Why is plasmid linearization important before transformation in recombineering experiments?

Answer 21

It ensures that the recombined DNA is correctly processed and integrated into the bacterial genome.

Question 22

What is genetic complementation, and how does it help confirm knockout phenotypes?

Answer 22

It serves as a control to ensure that any observed phenotype is due solely to the gene deletion. It involves reintroducing a functional copy of a gene to confirm that observed phenotypic changes are due to its deletion.

Question 23

Which bacterial species are commonly used in recombineering-based pathogenesis studies?

Answer 23

Neisseria meningitidis, Haemophilus influenzae, and Moraxella catarrhalis.

Question 24

How is recombineering used in bacterial pathogenesis and vaccine development?

Answer 24

Alters surface proteins for immune interaction studies Helps identify potential vaccine antigens

Question 25

What role do CEACAM receptors play in bacterial pathogenesis?

Answer 25

They serve as binding sites for pathogenic bacteria, facilitating adhesion and infection.

Question 26

What are some challenges faced when using recombineering in bacterial genetics?

Answer 26

Off-target recombination, low recombination efficiency, and unintended mutations affecting nearby genes.

Question 27

How does recombineering enable controlled gene expression in bacteria?

Answer 27

By inserting regulatory elements, researchers can control when and how a gene is expressed.

Question 28

How can successful recombineering and its phenotypic effects be validated?

Answer 28

PCR screening and protein detection methods such as Western blot or ELISA. PCR, Western blotting, transcriptomics, and functional assays to confirm gene expression and phenotypic effects.

Question 29

How can recombineering be applied to study protein function?

Answer 29

It enables the tagging, overexpression, or deletion of genes to study their role in bacterial metabolism and pathogenesis.

Question 30

What are potential future applications of recombineering in microbiology research?

Answer 30

Synthetic biology, antibiotic resistance studies, and engineering bacteria for therapeutic use.

Question 31

How can recombineering help investigate specific virulence factors like adhesins or immune evasion proteins?

Answer 31

Enables targeted removal or modification of adhesin or immune-evasion genes. Allows testing of protein function through domain-specific mutagenesis. Facilitates complementation studies to confirm phenotypic effects. Supports expression of altered versions to assess host interaction outcomes.

Question 31

How can recombineering be used to study bacterial virulence and host interactions?

Answer 31

Allows precise mutation or deletion of virulence genes. Enables construction of reporter strains to monitor infection dynamics. Facilitates knockout/knock-in of genes to study roles in immune evasion and adhesion. Helps dissect how bacteria interact with host cells at the molecular level.

Question 32

Why is matching expression levels important in recombinant protein studies?

Answer 32

To ensure functional studies reflect natural biology. Over- or under-expression can alter function, mislead results, or cause mislocalization.

Question 33

What happens if too much recombinant protein is made in a bacterial cell?

Answer 33

It may misfold, not be processed properly, and accumulate in the cytoplasm, preventing proper localization and function.

Question 34

Why is tropism important in protein expression studies?

Answer 34

It ensures proteins are expressed in the correct cellular location, which is essential for their function.

Question 35

Why was there ethical controversy around gain-of-function influenza research?

Answer 35

Researchers engineered more virulent strains, raising fears about potential pandemics if released or misused.

Question 36

What legal/ethical frameworks govern genetic modification experiments?

Answer 36

You need specific licenses to modify organisms; experiments must comply with strict containment and regulatory standards.

Question 37

What is the advantage of expressing and studying recombinant proteins outside a pathogen?

Answer 37

It avoids infection risks and allows focused biochemical and structural analysis, e.g. ligand-receptor interaction.

Question 38

What must be removed when cloning eukaryotic genes for expression in bacteria?

Answer 38

Introns must be removed; only exons (via cDNA) should be used to ensure proper expression.

Question 39

What are common cloning methods for protein expression?

Answer 39

Restriction enzyme-based cloning and Gateway cloning (recombinase-based) are commonly used.

Question 40

How can recombinant proteins be purified without a tag?

Answer 40

Via sequential chromatography based on ionic charge, hydrophobicity, and size.

Question 41

What is a His-tag and how does it aid protein purification?

Answer 41

A 6-histidine tag binds strongly to nickel columns, allowing selective purification of the tagged protein.

Question 42

What is the benefit of an immunoglobulin Fc tag in protein expression?

Answer 42

It facilitates secretion and allows purification using protein A/G that bind Fc regions.

Question 43

What do protein A and protein G bind to?

Answer 43

The Fc region of antibodies; used to purify Fc-tagged proteins or antibodies.

Question 44

What are CEACAMs and why are they relevant to host-pathogen studies?

Answer 44

Carcinoembryonic antigen-related cell adhesion molecules; receptors for many human-restricted pathogens.

Question 45

Which pathogens studied bind CEACAMs and where are they typically found?

Answer 45

*Neisseria*, *Haemophilus influenzae*, and *Moraxella catarrhalis*; all colonize the nasopharynx.

Question 46

Which CEACAM receptors are widely vs. narrowly expressed?

Answer 46

CEACAM1 is widespread; CEACAM3 is neutrophil-restricted; CEA and CEACAM6 are in mucosal epithelium.

Question 47

How do *Neisseria* and *Haemophilus* bind CEACAMs?

Answer 47

Via transmembrane beta-barrel proteins with surface-exposed loops.

Question 48

What loops in the *Neisseria* opacity proteins bind CEACAM?

Answer 48

Loops 2 and 3 form the binding cleft.

Question 49

Which loops in *Haemophilus* P5 are involved in CEACAM binding?

Answer 49

Loops 1 and 3.

Question 50

How does *Moraxella catarrhalis* bind CEACAM1?

Answer 50

Using a trimeric autotransporter adhesin, UspA1, with a long coiled-coil stalk and head domain.

Question 51

What was the key domain in UspA1 responsible for CEACAM1 binding?

Answer 51

The RD7 fragment in the stalk, not the head as expected.

Question 52

How was the RD7 domain shown to bind CEACAM1?

Answer 52

Through recombinant fragment testing and confirmation of binding via Western blot and ELISA.

Question 53

What is convergent evolution in the context of CEACAM binding?

Answer 53

Different pathogens evolved distinct proteins to bind the same region of CEACAM1.

Question 54

How was the essential CEACAM1 binding site confirmed?

Answer 54

Mutating key residues like I91 on CEACAM1 abrogated binding across all tested species and proteins.

Question 55

How was the RD7 binding motif narrowed down?

Answer 55

By truncation and mutagenesis, revealing a ~15-aa pocket responsible for binding.

Question 56

Why is it important to test multiple bacterial strains in functional studies?

Answer 56

Some strains, including reference strains like 035E, may lack critical motifs (e.g. RD7) and give misleading results.