Introduction to:

Bacteriophage Genetics

http://hiv.sourceforge.net/launch/

2

Why ?

1. Bacteriophages are the bacterial equivalent to Viruses – we have learned a lot on Viral mechanisms from phages.

2. Handling of phages is similar to handling of Viruses – just simpler!

3. Viruses causes immediately life threatening diseases like SARS and EBOLA, chronically diseases like AIDS and cancer and everyday diseases like common cold and influenza.

4. Studies on phages was the single most important contributions to development of the “Molecular Biology”.

3

Reports

Deadlines:

2 weeks after completion: Tuesday April 8’th 2008.

No late reports will be accepted!Questions: Tuesday April 1’st 13.15 – 15.00 in my office in 16.2

Reports are delivered in my letter box: Ole Skovgaard in 18.1

I must have corrected all reports before May 16’th 2008 - and you turn in corrected reports in due time that I can fulfill this!

4

Viral life cycle, simple version:

Picture from Brock 10’th Ed.

5

Comparison of virus with cells:

Cell: Phage/Virus:

Protein Protein

DNA DNA or RNA

RNA

Lipider (Lipider)

Cellwall

components

-

Phages / Viruses feed on cells!

Phage receptors: lamB (malB)T6 tsxT1 tonB

6

Examples of different Coliphages

R.G. Glass: Gene Function, Crohm Helm 1982 London

PhageSize(nm)

Geno-me

Size(kb or kbp)

60DS lin DNA

49

T480x 100

DS lin DNA

166

T5 60DS lin DNA

110

T7 58DS lin DNA

40

X174 25SS circ DNA

5.4

FdSS circ DNA

~6

MS2 26SS lin RNA

3.6

7

Plating

Plating: Pictures from Brock 10’th Ed.

8

I: Genetic Recombination

Genetic distance: Distance %

Cotransduktion %

Centimorgan

Minutes

Bp

A

B

A B

A B

9

II: Genetic complementation

cistron A cistron B

cistron A cistron B

No complementation

cistron A cistron B

cistron A cistron B

Complementation

Figure 6.4: Cis-trans test.

Drying of plates before spot tests: 10' @ 37 °C with fan.

10

III: Induction of lysogenic bacteria

Figure 6.5: Lytic and lysogenic cycle (Modified from: A. Lwoff: "Lysogeny" Bacterial Rev. 17:269-337 (1953) .

11

III: Induction of lysogenic bacteria

PE: Establishing lysogeny; requires cII PM: Maintains lysogeny, autoregulated by cIPL and PR: major early promoters of lysis

The cI857 protein is temperature sensitive:Active @ 30°C and inactive 42°C

12

IV: Restriction / Modification

MM MM

MM

MM

MM

MM

MM

MM

13

How to work efficiently in the lab:

Object:

Phage techniques and classical experiments.

Management of several ongoing parallel experiments: Before starting an experiment: Study the manual.

Compare text and flow diagrams. Make sure to understand the reason for each step.

Before leaving an experiment: Make sure you have the following for the next step(s):

• Knowledge: what to do and why.

• Materials: what you need and ready to use. And then you can do the other experiment or have a

break!

14

Notes for the statistics:

(3) %1002

progenytotal

progenyrIInceDista

You have this formula:

Of which type is that?

oraora xxxx 2121

?2

121 x

xxx aora

15

Notes for the statistics:

For this formula:

The standard deviation, , is calculated as:

xxa

2

1

xxa

a

2

2

1

122

16

Notes for the statistics:

How can you estimate ?

Theoretical: for a Poisson distribution is given by:

Estimate from the observed standard error, SE:

1

)( 2

nSE

xx

2

17

Notes:

Practical:

Phages and cultures are place on your bench top.

Phages and cultures are diluted and ready for use.

Inspection of plates: Tuesday @ 13:00.

Questions for the report:

While reading the plates.

Tuesday, April 1’st 13.15 – 15.00 in my office in 16.2.

Safety:

Chloroform & chloroform waste stays in the fume hood.

Other?