Bacterial Genetics Flashcards
What is genomics?
The study of genomes and DNA/RNA, genome replication, gene expression, genetic variation and distribution
What are the two main components of the bacterial genome?
Chromosome and mobile genetic elements (like plasmids or prophages)
What is a plasmid?
Autonomously replicating circular DNA
What is a Prophage?
Viruses integrated into the chromosome
What can you do from a whole genome sequence?
Predict cell function. You can identify patterns and homologous to known genes and motifs
Where are the most differences between different strains of genomes found?
In the MGE
What is a single nucleotide polymorphism?
Error in DNA replication
What do many Mobile Genetic Elements encode?
Virulence, antimicrobial resistance or host specific genes
What can acquisition of MGEs lead to?
New bacterial variants with enhanced virulence or resistance or host range
What are the three major pathways for the MGEs to move between bacteria?
Bacterial transformation, transduction and conjugation
How does bacterial transformation work?
Bacteria dies and releases the viral DNA into the environment. The recipient cell takes up that DNA up from the environment
How does bacterial transduction work?
Bacteria is either infected with a virus, or the virus in the genome is popped out (usually under stressful conditions)
Bacteria then undergoes the lytic pathway and lysed releasing the virus
Virus then binds to a new bacteria and injects their genes into the recipient
How does bacterial conjugation work?
Plasmids encode for the conjugation ability. Two bacteria come into contact with one another and make a pore between the two that allows the plasmid to move across to the other bacteria
What is a lytic pathway in bacterial genomics?
When a virus starts to dictate what happens in the bacteria, so it makes lots of copies of itself
Give some features of mobile genetic elements
- autonomously replicating circular DNA - not essential to the host bacteria - easy to manipulate
What size are mobile genomic elements?
2 kb to >100 kb
What is the restriction site on a plasmid?
Where you can add DNA
What is generalised transduction?
Temperate bacteriophages accidentally packages host bacterial DNA or plasmids into long phage particles and delivers it to new bacteria
Can all DNA transfer into any bacteria?
No
What does the restriction modification enzyme do?
Binds to a specific sequence on the DNA and digests it
How many subunits do restriction modification enzymes have?
3
How do bacteria protect themselves from self digestion?
Makes a copy of the restriction modification enzyme without the enzyme bit, which methylates that bit of DNA, marking it as ‘self’
What are some environmental triggers for bacteria?
Nutrients, oxygen, iron, temperature, bacterial pheromones, mammalian cells / hormones
What are bacterial pheromones used for?
How the bacteria speak to each other
Where are many bacterial virulence factors soley expressed?
In Vivo or in conditions mimicking those found in Vivo
Where is ‘in vivo ‘?
In the host(the body)
What can you do in transcriptional genomics?
Change the conditions depending on what you’re interested in
What are the steps in transcriptional genomics?
- grow bacteria in conditions of interest - extract bacteria - extract bacterial mRNA - convert mRNA to DNA - sequence the DNA and quantitate the numbers of transcripts of each genes
Why do we manipulate bacterial genomes?
To make tools for industrial production of proteins and for studying bacteria or gene function
What is the first step in cloning?
Cut two different pieces of DNA using a restriction enzyme, leaving matching sticky ends on both . Mix the two together and use lipase to seal the join
What are the most common cloning vectors?
Plasmids
What is a cloning region?
Target site for multiple restriction enzymes
Can any DNA be cloned?
Yes
How do you check which clones have worked correctly?
Culture the cells in conditions where only the gene you’ve taken up could grow (add the antibiotic if you’ve added an antibiotic resistant gene)
How do you construct a knockout (5)?
1- clone virulence gene into ‘suicide vector’ plasmid
2- clone antibiotic resistance marker into the gene to disrupt it
3- transform into a bacterial cell
4- Recombination via RecA protein (rare)
5- place onto agar with antibiotic and select for the rare isolate that has the resistance marker
What is CRISPR?
Genetic manipulation of eukaryotes
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
What is a knockout?
When one of the organisms genes are made inoperative (knocked out)
