Lecture 4: Bacterial Genetics and Gene Transfer 2

Question 1

What is an ORF (open reading frame) and how many do bacterial genomes encode (approx)

Answer 1

ORFs is the DNA sequence between the start and stop codons, thus, the translated portion of DNA.

There is ~100ORFs per 1Mbp - each ORF ~1000bp

Question 2

What does ‘Core Genome’ mean?

Answer 2

Genome shared by all strains of a given species

Question 3

What does ‘Pan Genome’ mean?

Answer 3

Genome including core genome + genes not shared by all strains of a given species. Genome outside of the ‘core’ is typically accessory functions.

Question 4

What is meant by ‘Mobilome’

Answer 4

“Sum total of all mobile genetic elements (MBEs) in a genome”

Includes: plasmids, prophages, other (integrons, insertion sequences, transposons)

Question 5

What is a Bacterial Defence System and what are some examples?

Answer 5

Weapons against exogenous DNA attack (typically phage DNA)

1. Restriction modification system (restriction enzymes)
2. CRISPR Interface

Question 6

Explain the Restriction Modification System and its two main parts

Answer 6

1. Restriction Process: Destroying DS-exogenous DNA at specific sites using restriction endonucleases (RE)
2. Modification Process: Host protecting itself from restriction enzyme attack, done via modifications, typically methylation of RE target sequences

Question 7

How long are typical recognition sequences for restriction enzymes

Answer 7

typically palindromes of 4-10bp long

Question 8

What is a palindrome?

Answer 8

ANYTHING LIFE THAT IS THE SAME FORWARD OR BACK, e.g., racecar

In this case, DNA, e.g., ->GAATTC->
(EcoRI)

Question 9

What is molecular cloning?

Answer 9

Movement of a desired gene or DNA fragment from its original source to a small genetic element (vector). Results in RECOMBINANT DNA

Question 10

What is the process/mechanism of molecular cloning?

Answer 10

1. Using restriction enzymes, cut the DNA source into the “insert” and vector to linearize it
2. ligase the DNA insert and linearized vector
3. introduce recombinant vector into a host

Question 11

What prokaryotic organisms is CRISPR present?

Answer 11

Bacteria and archaea

Question 12

What is the CRISPR array and what is it composed of?

Answer 12

The CRISPR Array is essentially the library of foreign DNA. It is composed of unique foreign DNA sequences called SPACERS separated by identical REPETATIVE SEQUENCES

Question 13

How does CRISPR work?

Answer 13

CRISPR region is transcribed into a long RNA molecule. Long transcript then processed into segments by CRISPR associated (Cas) proteins, this converts the sequences into segments of small RNAs called CRISPR RNAs (crRNAs).

A crRNA base-pairs with an invading nucleic acid, which is then destroyed by the endonuclease activity of a Cas protein.

Question 14

Give examples of CRISPRs potential applications

Answer 14

1. Genome editing
   - Creating specific cuts in DNA sequence of virtually any cell - for this, SYNTHETIC GUIDE RNA (sgRNA) is designed to target specific sequence.
   - Genes for sgRNA and Cas9 often cloned into a plasmid to be delivered