Hosts
- bacteria (E. coli)
- yeast (S. cerevisiae)
- insects (D. melanogaster)
- plants (Arabadopsis thaliana)
- mammals (Mus musculus)
- a cloning host must:
- be easy to handle and propagate
- have a defined genotype
- accept a range of vectors
- bacteria are simple, but do not have the capacity for eukaryotic post-transcriptional or post-translational processing
Cloning vectors (Image)
- plasmids
- bacteriophage and derivatives
- artificial chromosomes
- transposable elements
- plasmid and other cloning vectors (e.g pUC19) must have the following characteristics:
- small size (2.69 kb)
- origin of replication (at position 900 bp on the map)
- selectable markers (lacZ and ampR genes)
- unique cloning sites (polylinker)
- use a media that contains ampicillin and X-gal
- X-gal is a lactose analog that, when modified by B-galactosidase, turns the bacterial colony blue
-the polylinker is interior to the lacZ gene
- separates the promoter from the coding region
- if a bacteria receives
- no plasmid: AmpS and lacZ-
- no growth
- intact plasmid: AmpR and lacZ+ (Image)
- blue colony
- recombinant plasmid: AmpR and lacZ-
- white colony
- recombinant plasmids are restricted to about 7-8 kbp in size
- bacteriophage
- lytic/lysogenic
- lambda, T4, M13
- 15 kb of the lambda-genome involved in lysogeny (Image)
- not essential for cloning
- packaging of lambda-DNA is size sensitive
- must be between 45 and 51 kb
- delete non-essential -DNA, replace it with insert DNA
- Charon 40 (Image)
- stuffer fragment, keeps size above minimum for propagation
- cut with enzyme
- swamp with foreign DNA
- 2 arms ligate, non-viable
- viable phages will be recombinant with a piece of foreign DNA (Image)
Cosmids
- lambda-DNA replicates via rolling circle replication
- "cos" sites are regularly spaced (Image)
- genome is snipped
- small plasmid with a cos site
- linearize and mix with foreign DNA
- ligate 2 cosmids on appropriately sized DNA (32-47 kb)
- will package into phage heads
- phage is infectious, injects into E. coli (Image)
- Yeast Artificial Chromosome
- good for cloning large pieces of DNA (up to 500 kb)
- bacterial ori, Ampr, yeast ori, centromere, two telomere sequences, selectable markers (Image)
- grow YAC in bacteria, purify
- cut with two restriction enzymes
- creates two "arms"
- add foreign DNA, ligate
- two arms on either side of insert (Image)
- transform auxotrophic yeast
- trp1- and ura3-
- properly ligated (by chance) YAC will be Trp+ and Ura+
- will stably harbour the large piece of DNA
- entire eukaryotic gene (introns and everything!)
- artificial chromosomes for bacteria (BAC) and mammalian (MAC) systems (Image)
- Chromos Molecular Systems
p-element derived vectors
- transposable element in Drosophila
- P strain fly has lots of p-elements in the genome
- M strain fly has no p-elements
- P-sperm and M-egg, very few progeny
- p-elements jump around in germ line
- hybrid dysgenesis
- M-sperm and P-egg, normal
- Why?
- p-element requires enzyme "transposase"
- p-strain cytoplasm also has a high level of a transposase repressor
- p-sperm contributes little cytoplasm
- repressor diluted
- transposition
- vector has inverted repeats, and polylinker (Image)
- helper element has transposase gene
- cannot itself transpose
- inject both into Drosophila embryo (Image)
- P-element will insert into chromosome, taking the foreign DNA with it
- can insert up to 40 kb
plant cells
- Agrobacterium plasmid delivery system
- Ti-plasmid transfers from soil bacterium to dicot plants
- T-DNA ('transferred'-DNA) contains genes for tumorous growth
- integrates into host chromosome
- vir genes mediate infectivity (virulence)
- split the Ti-plasmid into two (Image)
- one contains the vir genes
- other contains the T-DNA, with most deleted
- the flanking ends are retained, as these are what are directed to be transferred
- antibiotic resistance gene for transformant selection
- vir genes direct the T-DNA deletion plasmid into the new host (Image)
