Hunting the gene L7

Question 1

How did Benzer go about answering the question of how many genes the r11 region had?

Answer 1

A complementation test: when E.Coli K is infected by 2 different r11 mutants separately a plaque only forms if mutations are in different genes
Mutations in same gene fail to complement

Question 2

How many genes did Benzer find?

Answer 2

2 because r11 mutations fell into 2 complementing groups, r11A and r11B genes
(see notes for diagram)

Question 3

How do bacteria fight back against phages?

Answer 3

Use CRISPR and encode restriction endonucleases which recognise specific base sequences in the phage chromosome and introduce double-strand breaks eg. EcoR1 , Sma1

Question 4

What are type 11 restriction enzymes?

Answer 4

Typically have recognition sequence 4-10 bp which is palindromic ie. reads same on both sides of the double helix

Question 5

What are flushed and staggered ends?

Answer 5

see notes

Question 6

How many restriction enzymes are commercially available?

Answer 6

over 100

Question 7

How do bacteria guard against cleaving their own DNA?

Answer 7

Encode a sequence-specific methylase enzyme that chemically modifies 1 base in the recognition sequence to make it immune to its own restriction enzyme (prokaryotic epigenetics!)

Question 8

What is generalised transduction?

Answer 8

At low frequency of 10^-4, fragment of bacterial chromosome is packaged into phage particle, bacterium can incorporate new DNA after infection into its chromosome by recombination

Question 9

What is conjugation?

Answer 9

see notes for diagram
Done with plasmids of a few kb–> 100kb
Best known to happen with plasmid F where a new copy of F generated by replication is transferred to a recipient cell
Plasmid F can integrate into E coli chromosome via homologous recombination

Question 10

What is transformation?

Answer 10

Bacteria take up DNA from surroundings and incorporate into own genome (see notes for diagram)
seen in studies some bacteria actively attack related organisms causing lysis and take up released DNA by transformation

Question 11

How can you treat e.coli in lab to make transformation efficient?

Answer 11

CaCl2

Question 12

What are multicopy plasmid (eg. ColE1) genome size and how many copies per bacterial cell?

Answer 12

10 kb

40-60

Question 13

What happened in 1973 for the first time?

Answer 13

Hybrid plasmid made by cutting 2 plasmids with restriction endonuclease and rejoining fragments with DNA ligase

Question 14

Why are recombinant plasmids useful? Draw diagram of recombinant plasmid

Answer 14

Gives way to insert gene of animal origin into bacterial plasmid, introduce by transformation into bacterial host and make millions of copies (see notes for diagram)

Question 15

What 3 features do plasmid cloning vectors need?

Answer 15

1) replication origin so it can be copied into host
2) selectable marker so host transformation can be seen eg. ampicillin resistance gene
3) (useful not essential) insertion of foreign DNA fragment disrupts gene and produces recognisable phenotype eg. tetracycline resistance gene

Question 16

What is the limit of using plasmids as a cloning vector?

Answer 16

if over 20kb hard to take up by tranformation (10kb is a push)

Question 17

What method should be used for cloning DNA fragments 10-15 kb?

Answer 17

Phage lambda, central 15kb of genome not needed so can be cut out by restriction enzymes and replaced by foreign DNA

Question 18

What are steps for gene library construction?

Answer 18

1) DNA partially digested by frequently cutting enzyme(digestion with only partial fragments should virtually represent whole genome)
2) Use electrophoresis to isolate fragments ~ 15kb
3) Fragments ligated randomly into phage chromosomes from which central portion was removed by BamH1 digestion
4) Pack lambda chromosomes into long concatenates via sticky ends (see notes for diagram)
5) Package long concatenates into phage heads in vivo
6) Cloned sequences amplified by round of infection, collection of plaques form library of genomic DNA where you can potentially find any gene from organism

Question 19

How many base pairs can bacterial artificial chromosomes clone?

Answer 19

100kb+

Question 20

What are YACs

Answer 20

Have components of eukaryotic chromosomes and selecable marker, can accept up to 1000kb

Question 21

What is a cosmid?

Answer 21

A plasmid-phage hybrid that has a specific site (cos) allowing it to be packaged efficiently by phage proteins, can clone up to 40kb

Question 22

What is a strain where plasmid F has integrated into the chromosome called and what does the integrated F do?

Answer 22

Called HFr
Integrated F still capable of cell to cell transfer but takes whole chromosome with it
If F reemerges from HFr strain it sometimes carries the part of the chromosome adjacent to the integration site eg. F’lac carries lac genes