Learning Outcomes

At the end of this lecture, students will be able to: Explain the structure and functioning of M13 phage particularly in regard to replication mechanisms. Describe the use of M13 phage vectors in genetic engineering

M13 a filamentous phage

-completely different in structure from -circular single stranded DNA -6400 base pairs long -packaged inside rod-shaped protein capsid -The genome codes for a total of 10 genes (named using Roman numerals I through X) - fewer genes and simpler infection cycle

Gene VIII codes for the major structural protein of the bacteriophage particles
Gene III codes for the minor coat protein The gene VIII protein forms a tubular array of approx. 2,700 identical subunits surrounding the viral genome Approximately five to eight copies of the gene III protein are located at the ends of the filamentous phage (i.e. genome plus gene VIII assembly) Allows binding to bacterial "sex" pilus during sexual conjugation Pilus is a bacterial surface structure of E. coli which harbor the "F factor" extrachromosomal element (plasmid)

Infection
Single strand genome (designated '+' strand) attached to pilus enters host cell Major coat protein (gene VIII) stripped off

Minor coat protein (gene III) remains attached pilot protein controls the insertion of DNA into cell. Host components convert single strand (+) genome to double stranded circular DNA (called the replicative or "RF" form) Transcription begins -Series of promoters -Provides a gradient of transcription such that gene nearest the two transcription terminators are transcribed the most -Two terminators One at the end of gene VIII One at the end of gene IV Transcription of all 10 genes proceeds in same direction

Amplification of viral genome

Gene II protein introduces 'nick' in (+) strand Pol I extends the (+) strand using strand displacement (and the '-' strand as template) After one trip around the genome the gene II protein nicks again to release a completed (linear) '+' genome Linear (+) genome is circularized During first 15-20 minutes of DNA replication the progeny (+) strands are converted to double stranded (RF) form These serve as additional templates for further transcription When enough RF is made, Gene V protein binds to the (+) viral strand prevents synthesis of complementary strand only single stranded (+) viral DNA is build up (M13 genome)

Phage packaging
The ssDNA is encapsulated as it passes through the cell wall M13 (+) genome, covered in ss binding protein - Gene V protein, move to cell membrane Gene V protein stripped off and the major coat protein (Gene VIII) covers phage DNA as it is extruded out Packaging process is therefore not linked to any size constraint of the M13 genome Length of the filamentous phage is determined by size of the DNA in the genome Inserts of up 42 Kb have been introduced into M13 genome and packaged (7x genome size) ~8 copies of the Gene III protein are attached at the end of the extruded genome

- the bacteria is not killed but grow at a slower pace than uninfected cells gives turbid plaques ( not clear). - Recovery of phage particles from culture use polyethylene glycol. (First pellet the infected cells,

precipitate the phage from the supernatant with polyethylene glycol, extract the DNA with phenol and finally precipitate the DNA with alcohol )

- Double stranded RF DNA can be isolated and handled exactly like a plasmid (similar to construction of recombinant molecules). - single-stranded DNA can be prepared by removing virus protein coat.

Use of single-stranded vectors

-Filamentous phages (ssDNA) can be used for cloning &
amplification. -Advantages: it can replicate via RF and therefore can be manipulated in vitro like a plasmid both RF and ssDNA forms can transfect competent E. coli to yield either plaques/infected cells depends on type of assay used. No constraints of having to package recombinant DNA. Viral DNA up to 6x size of M13 has been cloned. It is easy to determine the orientation of the insert detection on agarose gel.

Development of filamentous phage vector

- do not have any non-essential genes for cloning sites - in M13 however, there is a 507 base pair of intergenic region (from 5498-6005 of the DNA sequence) which contains the origin of replication for both the viral and complementary strands. -DNA can be inserted into this site (intergenic region) -also possible to clone at the carboxyl-terminal end of gene IV -wild type M13 only has few sites (RE) within intergenic region.

M13 Family Tree (Development)

M13 (has 10 BsuI sites in genome) Isolate linear fragment (full length)

Blunt ended (Mung Bean/SI nuclease)

Ligated to Hind III (blunt end) fragment containing E. coli regulatory region with genetic information of -peptide of -galactosidase

Transform into E. coli strain with a deletion of -galactosidase -fragment

Recombinant M13 detected by intragenic complementation on X-gal + IPTG media

Blue plaque (complementation occurs) M13mpl

--- Insertion of DNA fragments into the lac region of M13mpl destroys its ability to form blue plaque selection for recombinants.

- -Gronenborn and Messing introduced an EcoRI site into lac region of M13mpl M13mp2
- - Messing et al. (1981) constructed M13mp7 which has a multiple cloning site in the lac region

a gene for the lac repressor (lac I) protein to allow regulation of the lac promoter the operator-proximal region of the lac Z gene (to allow for a-complementation in a host with operator-proximal deletion of the lac Z gene). a lac promoter upstream of the lac Z gene a polylinker (multiple cloning site) region inserted several codons into the lac Z gene