MICB325 Lecture 16: Reverse Genetics

• MICB325 Analysis of Microbial Genes and Genomes explores gene function with reverse genetics in Lecture 16

Reverse Genetics

• A gene-centric approach is utilized to investigate a specific gene's role in a biological process
• The process involves mutating a specific gene and observing the effects on phenotypic outcomes
• It tests a hypothesis, such as identifying E. coli's che gene homologs and testing their requirement in chemotaxis through intentional mutation

Targeted Mutagenesis

• Differs from random mutagenesis methods as it aims at specific genes of interest
• Used to evaluate genes in an operon, identify genes with predicted importance based on similar genes' roles, and determine the role of a domain or residue within a protein
• Natural homologous recombination mechanisms enables it in microorganisms

Natural Phenomenon

• Repairs broken DNA fragments during replication
• Requires a few hundred bp of nearly identical sequence
• Facilitates genome adaptation and evolution

Rec Proteins

• RecBCD cuts DNA strand at Chi sequence
• Resulting ssDNA is coated with RecA
• RecA invades another double-stranded DNA and nucleoprotein, scanning for homology
• A second nick occurs, and strands are exchanged

Homologous Recombination

• Occurs at identical sequences which results in a "cross over event"
• Foreign DNA can be incorporated into the genome, enabling targeted mutagenesis
• Typically involves many more base pairs up to and exceeding 200 bp

Plasmid Integration

• Plasmids integrate into the genome at sites where sequence identity, region of homology, exists
• A single recombination event results in integration

Multiple Recombination Events

• Can result in the exchange between the plasmid and the chromosome
• Exchanged fragments sit between "regions of homology"
• Can be used to insert foreign sequences into genome in a process referred to as genome editing

Replacing a Gene vs a Marker

• The plasmid should include regions of sequence identity flanking the gene and antibiotic resistance marker
• Create the desired sequence in the genome by using your plasmid
• Use a suicide vector, as marker is not maintained in cell unless recombination occurs

Replacing a Gene vs a Selection Marker

• This process requires selection for two successive crossover events, based on Amp resistance and sucrose sensitivity
• SacB performs a counter selection for sucrose

Making a Clean Deletion

• Requires 2 regions of homology with nothing in between on plasmid
• Is Amp sensitive and sucrose resistant like the original genotype
• Step 1: select single crossover species, which must be antibiotic-resistant and sucrose-sensitive to grow and propagate
• Step 2: counter-select on sucrose
• Selects for second crossover, double recombination, which are antibiotic-sensitive and sucrose-resistant like WT

PCR Screening

• PCR screening distinguishes possibilities to always check the final constructs

Applications of Targeted Mutagenesis

• Wild-Type
• Deletion that is precise and in-frame for either the portion or the entire gene
• Replacement with marker or label
• Mutation of WT gene by substitution of residue or domain
• Insertion of a label

Clean Deletions

• Clean deletions minimize polar effects

Generating a Point Mutation

• Introduction of a point mutation into a plasmid provides a mechanism to perform site directed mutagenesis

Targeted Mutagenesis: Step-by-Step

• Isolate DNA from wild type bacteria
• Amplify ~500 bp regions flanking the region you want to delete
• Clone these into a suicide vector flanking sequence to exchange
• Conjugate plasmid into wild type bacteria
• Select for antibiotic, vector can't replicate, only get AbxR cells if plasmid integrates into genome
• Then, select sucrose resistance, using sacB counter selection
• Check final construct using PCR

Summary of Targeted Mutagenesis

• Reverse genetics relies on this process
• Utilizes homologous recombination which exchanges DNA between two homologous regions, regions with the same sequence.
• Gene insertions and deletions require consecutive steps of selection and counter-selection

Deleting Genes

• Gene essentiality depends on the identification method
• Many gene products are only required under specific conditions
• Deleting an essential gene prevents growth/kills bacteria

Essential Genes in Pathogens

• A gene encodes a function required for bacterial survival during infection.
• Represents complement of possible antibiotic targets for possible drug discovery.

Testing for Essentiality

• If a gene is essential, it can't be deleted by homologous recombination b/c all colonies contain WT gene.
• Failure to remove gene in a large number of resolution reactions indicates a gene is essential.
• Absence of evidence is not evidence of absence
• Adding a second copy of targeted gene allows deletion only in the presence of the functional second copy
• Crossover frequency is used used to estimate probability that a gene is truly esssential

Integrative Vectors

• Used for stable integration into genome in single copy through non-homologous recombination
• Use an integrase to insert plasmid with attP site into chromosome, with attB site.
• Most bacterial genomes have an attB site in a non-essential gene.
• Features MCS, AprR, attP (phiC31), and int (phiC31)

Conditional Knockdown

• Achieved by introducing a meridiploid strain where the second gene copy is transcribed by a controlled promoter
• Tet-off system makes this possible, which prevents gene activation in presence of tetracycline or doxycycline
• Testing:
  • Delete WT gene in absence of tet, ie. second copy still expressed
  • Test viability with expression of concentrations of tet, reducing "essential" gene

Mycobacterium Tuberculosis (Mtb)

• The transcription termination factor Rho is a potential target for therapeutic intervention
• Inhibition of Rho prevents the growth of some bacteria
• The Rho protein of M. tuberculosis is essential for viability in a mouse infection model

Rho (p)-Dependent Termination

• Rho (p) helicase unwinds RNA:DNA hybrids and binds to the rut sequence (rho utilization)
• Catches up to the RNAP when the RNAP pauses helicase activity and unwinds DNA:RNA hybrid

Testing Rho Essentiality

• Under control of RevTetR, the Rho gene can be inserted in the att site of Mtb both with and without Arho, Strain A and B.
• In murine models Rho is shown to be essential.

Summary

• The inability to delete a gene using homologous recombination is evidence that it may be essential
• Essential genes can be deleted through the addition of a second copy of the gene under a repressible/inducible promoter