Molecular Genetic Screening in Bacteria Flashcards
What is a genetic screen?
What does a genetic screen tell you about the gene(s) identified?
Series of experiments designed to discover the function of specific genes/sets of genes
Genes identified are important for surviving the selection pressure applied
Process of a genetic screen? (3 steps)
What selection pressure will we use?
- How will this help identify cell division genes?
Make a mutagenised library
Apply selection pressure
Further study
Temperature selection pressure of 42°C causes bacteria to elongate as they cannot divide; Eventually elongate so much they kill themselves
- Strain with mutations in cell division genes will elongate if they have a temperature sensitive mutation
Process of genetic screen –> Identifying genotype? (hint - 5 steps)
- Hypothesis – Molecular genetic screening will identify new genes/proteins involved in these pathways
- Mutagenesis – Chemical mutagenesis
- Screen selection – Temperature 30°C - 42°C; Searching for filament temperature sensitive mutants
- Hit validation – Complementation and gene mapping
- Identify genotype
When using nitrosoguanadine to induce chemical mutation, we look for streptomycin resistance, which is caused by a known point mutation in ribosome of E. coli
What are we looking for to get 1 mutation in bacteria?
Looking for a sweet spot between getting enough cells with 1 mutation to study and not killing them
If you get 80 temperature sensitive (TS) mutants for every 10^8 cells, how many colonies would need to be screened for 10 TS mutants?
How long would this take?
Screen 12.5 million colonies
Would take years of time; Harsh reality of these screens
Go through Selection section of (5)
When do we stop screening?
When you find something new; Reality is you don’t have infinite time and funding
How do we know our 2/3 strains are not just ftsA ‘re-discoveries’?
Use complementation to determine if the phenotype is associated with ftsA or a new gene
- Remove gene, observe phenotype, put gene back in and see if that phenotype disappears
How does λ phage complementation work?
What is the B region?
Enters E. coli genome through recombination of attP and attB regions
B region is not essential for phage growth and can be deleted and replaced with DNA/gene insert of interest
Go through phage complementation in (5)
What does graph in (5) - identify genotype show?
What does this confirm?
At 42°C, the cfu is static but the absorbance is still increasing; Cells are growing and not dividing
Confirms elongation phenotype
Why are old bacterial genomes marked with minutes?
By putting conjugation plasmid in donor, they would transfer entire genome to recipient through pilus
Time at which gene was transferred to recipient was the time it was assigned
Using conjugation data, where is the new fts gene found on the E. coli genome? ((5) - Gene mapping)
Between LeuC and NadC marker
How do we identify the new genes primary DNA sequence using λ16-2 phage? (hint - restriction enzymes)
λ16-2 phage can be used to ‘cure’ TS phenotype in PAT84; Gene of interest is somewhere in λ16-2 – Specifically the b region
Sequence the phage by fragmenting with restriction enzymes and ligating it and then seeing if it still complements our strain
Using the digestion data and complementation information available, identify the order of the genes in the λ16-2 phage (and therefore the E. coli genome); + indicates that it complements ((5) - last page)
MurC and dd1 (can’t tell order)
ftsA
New gene (PAT84)
envA
Summary of data analysis 1:
Hypothesis
Mutagenesis
Screen selection
Hit validation
Identify genotype
Hypothesis - Hypothesised there was more about bacterial cell division to discover
Mutagenesis - Used Nitrosoguanadine to generate temperature sensitive mutants
Screen ‘Selection’ - Selected filamentous temperature sensitive mutants out of these
Hit Validation - Used the A16-2 phage to prove the gene was not ftsA and therefore a new gene
Identify Genotype/Gene mapping - Used bacterial conjugation to map this new gene to the 2 min region of the E.coli genome
- Mapped the gene to a region downstream of ftsA and named it ftsZ
In the modern era, what can we identify now rather than just genotype?
Gene function
There are currently unidentified Class A PBP enzymes in S. pneumoniae (gram +ve)
In gram -ve bacteria, these are controlled by regulatory proteins in outer membrane, LpoA and LpoB; These 2 protein complexes are vital for cell growth and division
Why doesn’t this apply to gram +ve?
They have no outer membrane; What is controlling these enzymes?
What is meant when we say GeneA and geneB have synthetic lethal relationship?
Need at least one to encode for essential function and survive – Without both the cell dies
If 2 genes (geneA and geneB) have a synthetic lethal relationship, what genes will become essential if geneA is inactivated?
GeneB + Any genes required for function of GeneB
How does transposon mutagenesis work?
Transposon mutagenesis works by gene disruption which is predicted to inactivate the gene it jumps into through homologous recombination into the gene
- Inactivates through gene truncation
How do we pair transposon mutagenesis and next generation sequencing?
What is this process called?
Called Tn-seq
Map large numbers of transposons
How does Tn-seq work?
Saturate the genome with many transposons and have every possible insertion represented
To interpret ‘gaps’ in this profile, the transposon insertions must have happened and then been lost from the population because they were selected against (killed) - These genes are essential for what we are looking for
If transposon sequencing works by transposons being lost from a complex population, how big do you need your Tn-seq transposon library to be?
- S. pneumoniae genome size – 2,046,115 bp
- S. pneumoniae gene number – 2069
2,000,000 members (~1 per 1 bp) is best to ensure enough saturation of genome with transposons; 200,000 (~1 per 10 bp) is more realistic and you still get good saturation
How do we screen/select for synthetic lethal genes?
How do we measure the difference between these 2 profiles? (2 ways)
Looking for a profile that tolerates gene of interest insertions in WT, but can’t tolerate gene of interest insertions in the knockout
- Look for gaps
Read count ratio; (No. of Insertions in A)/(No. of Insertions in B) – Bigger is better (>1)
Statistical measure; Mann Whitney U test
What is a p-value in statistical analysis?
Probability that the null hypothesis is true (essentially what you’re seeing is due to random chance)
If the number of genes in the S. pneumoniae genome is 2069, how many genes do we reasonably know are false positives using a p-value of <0.05?; What does this mean?
What should we do instead?
103 (2069 x 0.05); We’d have to screen 103 genes to see if just 1 is correct - Too much time
Instead use a p value of <0.005
- 10.3 genes need screening
What is a divide by 0 error and what causes it?
What are it implications?
We are looking for scenarios with 0 insertions in a particular gene; Causes errors in read count calculation and algorithm doesn’t include it in dataset
Summarise the genetic screen steps in the modern day PBP1a and PBP2a screen we did (5 steps):
Hypothesis
Mutagenesis
Screen Selection
Hit Validation
Gene Function
Hypothesis - We hypothesised that there are unidentified protein required for PBP1a and PBP2a function
Mutagenesis - We used a transposon-based Tn-seq screen to identify genes which are synthetically lethal with pbp1a (and pbp2a)
Screen Selection - The screen identified 10 genes synthetic lethal with pbp1a and more data analysis identified 2 more giving 12 in total
Hit Validation - Used a conditional expression systems to confirm SPD_0876 + SPD_0878 were synthetic lethal pbp1a
Gene Function - Used microscopy data to show SPD_0876 (MacP) was required for pbp2a function in vivo
Explain the 2 diagrams in (6) - Hit validation
You have identified 3 genes of interest and tested each in a pbp1a conditional expression strain with zinc (lets us turn it on and off at will)
How can we work out how the cells die in this synthetically lethal scenario with zinc expressed pbp1a? (look at (6) - Gene Function if you need diagram)
How do we get quantification of microscopy data?
Deny the cells zinc and they’ll slowly die
In non-WT they shrink without zinc
Analyse image using software (e.g. MicrobeTracker)
Measure the area of 250 cells/condition
Given the microscopy quantification data in (6) - Gene Function, what is the genetic relationship between SPD_0876, pbp1a, pbp2a?
Why is cell area measured rather than volume?
SPD_0876 is not required for pbp1a function, but is required for pbp2a function
Use a t-test to prove changes in cell area
Cell area was measured instead of volume as we have a 2D data set, so we can’t infer volume
