1. Types of cloning vectors
1. plasmids (about 20kb)
2. Bacteriophage (bacterial viruses), 30-50kb inserts
3. Cosmids (35-50kb insert)
4. BACs
Use fertility F plasmid
75-300kb inserts possible
developed during the human genome project
5. YACs
Mimics yeast chromosome
Contains all regions for replication (yeast ori and centromere)
kb inserts poss.

2. Lytic or Lysogenic life style Virulent phage
Viral vectors
1. Bacteriophage Lambda
Virus of E.coli
Lytic or Lysogenic life style
Virulent phage
do not integrate their DNA into the host bacterium genome and they usually kill the host (lytic)
Temperate phage may integrate into the host genome causing lysogeny

3. )

6. Bacteriophage lambda vectors are commonly used for construction of genomic libraries
Packaging of bacteriophage lambda
Packaging of DNA into the head does not require a full length of wild type DNA. Length of between % can be packaged (37-53kb)
To get packaging need 2 cos ends one at each end.
Usually 50 kb apart and this ensures only 50kb gets packaged
If further than 53kb , no packaging
An enzyme at entrance to head recognises and cuts at cos site

8. During replication, the phage DNA is produced in a concatameric form, which is cleaved by appropriate endonucleases to allow packaging of a single genome within the phage capsid.
It was found that internal regions of the phage genome, which were not essential to phage replication, could be removed and replaced with DNA of interest.
This hybrid DNA could be efficiently packaged, and form an infective phage.
Advantages of this type of system vs plasmids like pBR322
The phage genome is able to package efficiently with DNA inserts as large as 20 Kb.
the packaged phage are highly infectious and infect E. coli at a much higher efficiency than plasmid transformation methods.

9. Bacteriophage Lambda vectors
Derivatives have been manipulated to have unique restriction enzyme sites
Some are insertion some are replacement vectors
Some have replacement areas flanked by cutting sites
40% of the wild type lambda is dispensible and can be replaced
e.g. of lambda cloning vector (see diagram)
lambda sequences have been devised with 2 Bam H sites flanking the insertion excision region
When cut with Bam get 3 segments
Left arm has info for heads and tails
Right arm has info for DNA replication and lysis
Middle fragment can be replaced with cloned DNA about 20kb long.

10. replacement vector replacement vectors replacement vectors
P2 bacteriophage is already inserted into the genome of E.coli. It prevents reconstituted lambda bacteriophage from growing. Note Insertion excision bit is removed and only the lytic cycle can be followed in the recombinant phage..
Replacement of lambda DNA containing the red and gam genes in the lambda EMBL3 genomic DNA cloning vector with BamHI compatible DNA inserts of ~10-20 kb, permits lytic growth of recombinant hage in E. coli strains containing the P2 bacteriophage lysogen.
Insertional cloning into the cI gene of the lambda-gt10 cDNA cloning vector (DNA inserts of ~1-5 kb) can be selected in hfl (high frequency of lysogeny) mutant strains of E. coli. In hflA strains of E. coli, expression of the lambda cII gene is elevated, resulting in transcriptional induction of the lambda cI repressor gene which promotes lysogeny. Disruption of the lambda cI coding sequence by DNA insertion into the unique EcoRI site of the lambda gt10 cDNA cloning vector, blocks the lysogenic pathway leading to cell lysis and plaque formation.

11. -Can not fit as much new foreign DNA in
Insertion Vector
-Can not fit as much new foreign DNA in

12. The limitation to using lambda insertion vectors is their small insert size capacity. The analysis of transducing phage suggested that as much as 40% of the wild-type genome is dispensible for lytic growth. However viral genomes only 60% of the wild-type length aren't packaged into viable phage particles. Therefore, in order to utilize the full carrying capacity of the lambda vector,
substitution phage vectors were developed. In order to maintain the vector as a viral stock it must replicate efficiently. To take advantage of the full carrying capacity (vector constitutes 60 % of recombinant genome length, the insert %), the vector sequence must carry a 'stuffer' fragment which can be easily removed and discarded. This stuffer is replaced by the foreign DNA fragments we wish to insert. The foreign DNA fragments substitute for the stuffer fragment.
Substitution vectors ( KB) carry larger inserts than insertion vectors ( KB). Wild-type lambda phage do not efficiently infect E coli lysogenic for an unrelated bacteriophage P2. Thus, wild-type phage are Sensitive to Ps Interference (spi). Phage genes responsible for the spi phenotype are carried on the stuffer fragment of the insertion vector. Removal of the stuffer and its replacement by a foreign DNA fragment renders the recombinant phage insensitive to P2 interference. Therefore we can selectively propage recombinant phage and eliminate nonrecombinants (carrying the stuffer fragment) by growing on an E coli host carrying a P2 lysogen. All of the progeny phage carry foreign DNA inserts because nonrecombinants cannot grow due to the P2 interference.
Presence of CI gene inhibits the lytic cycle.

13. In vitro packaging systems
Can be made
Purified empty heads, lambda DNA with foreign insert (50kb with cos ends), and tail assemblies in test tube.
Result in infective bacteriophage
Uses
Cloning pieces too big for plasmids
Generation of ds DNA

14. M13 , a single stranded filamentous phage
Phage DNA is packaged in the core of a helical particle
The length of particle is dependant on length of DNA
In all M13 preps the following occur
polyphage- more than one genome length
minphage genome length
maxiphage- genetically defective but more than one genome length
Molecular biologists use this to create cloning vectors
Can insert long stretches of DNA into non essential regions
No sharp cut off in length that can be packaged
Some decrease in efficiency of packaging with increasing length
10% longer not affected, 50% longer replicate more slowly

15. How M13 infects and reproduces infects through pili
Protein coat is stripped and ss DNA is converted to double stranded replicative form
DNA relicated by “rolling circle method”
New particles assembled
200 particles per infected cell per generation
M13 released without lysis
No lysis on bacterial lawn, generally do in liquid culture.
Uses
Cloning
Ss DNA for probes, sequencing
Phage display technology, M13 will produce foreign protein on surface as part of its protein coat, can use to generate specific antibodies
Life cycle. Filamentous phage attach to the F-pili of E. coli and inject single-stranded
circular DNA into the bacteria. The ssDNA is converted to a dsDNA form known as replicative
form (RF). The replicative form behaves as a plasmid within the bacteria and undergoes
multiple rounds of replication. It is this replicative form that is isolated and manipulated as a
cloning vector. Foreign DNA can be incorporated into cloning sites and bacteria transformed by
standard procedures. Structural genes are transcribed from the replicative form and the ssDNA
genome is produced from a special replication origin (usually called the f1 origin). The product
of gene II forms a nick in the initiation site of the
f1 replication origin. Host DNA polymerases
replicate the phage DNA starting at the nick and
displace one strand will progressing along the
template. When the polymerase completes the
circle, the gene II product cleaves the displaced
strand at the termination site within the replication
origin and the ssDNA is circularized.
The ssDNA is packaged at the membrane of the bacteria and the mature phage is
extruded without lysing the cell. Phage particles containing ssDNA are isolated from the culture
supernatant medium by differential centrifugation and the ssDNA is extracted from the phage
particles. The phage can also be plated on a bacterial lawn to produce plaques. However, these
plaques are 'fuzzy' since they are not formed as a result of cell lysis, but formed as a result of
diminished host cell growth.

16. Adeno virus
Human virus
Used to insert DNA into human cells
Gene therapy
Normally causes respiratory diseases and pink eye. Want virus to infect our cells but not cause illness

18. Cosmids
Cross between phage and plasmid
Circular ds DNA
Contain cos sites and can be packaged into phage heads
Carry more DNA than plasmid and can be maintained and manipulated as plasmids
Cloning using cosmids
Clone into vector as would with plasmid
Introduce DNA to cell as you would with phage
Propagate as you would a plasmid
Cosmids have about 5kb DNA and therefore can get ~ 33-48kb foreign DNA into a phage head.

19. Measurement of viral growth
Must grow virus on host cells to see anything. Can't grow virus without cells.
To quantify viruses, need some way to get flat surface of growing cells, allow virus-infected cells to spread radially where present = plaque.
In bacterial cells this is easy. Spread "lawn" of bacteria on plate, add diluted phage suspension or culture infected with phages. After 6-8 hours can see plaques in E. coli.

20. Bacteria have three mechanisms to accomplish genetic recombination (mixing up of DNA):
1. transformation -acquire new genes by taking up DNA molecules from their surroundings
2. conjugation -can transfer a portion of their chromosome to a recipient with which they are in direct contact usually via a plasmid
3. transduction- via Bacteriophages - in the process of assembling new virus particles, some host DNA may be incorporated in them

21. Bacterial artificial chromosomes (BAC):
The F factor plasmid has the ability to continue to function even when integrated into a complete bacterial chromosome.
Highly modified F plasmids have been generated that are capable of cloning very large inserts of up to 300,000 base pairs.
One feature is the incorporation of cut sites for restriction endonucleases with eight base cut sites.
Such endonucleases cut DNA less frequently and thus generate larger fragments for cloning.
Bacterial artificial chromosomes are sometimes introduced into their host cells by electroporation, which consists of a brief treatment with high voltage electric current that momentarily disrupts the cell membranes and facilitates entry of large DNA molecules.
Once in the cell, the BAC replicate like F plasmids.
Yeast Artificial Chromosomes (YAC):
a yeast artificial chromosome (YAC) contains
a yeast origin of replication a centromere,
a telomere at each end
a large inserted DNA sequence of up to about 500 kb
Prior to insertion of the foreign DNA, the essential components of the YAC are maintained in bacterial cells as circular plasmids.