1. Chapter 4 Recombinant DNA Technology

2. Plasmid Plasmids Self replicating Double-stranded
Mostly circular DNA (<1 kb~ > 500 kb)
Linear : Streptomyces, Borrelia burgdorferi
Replicon
Maintained in bacteria as independent extrachromosomal entities : own replication origin
Generally dispensable

3. Types of plasmids F plasmid (conjugative) R plasmid
Conjugation
F plasmid (conjugative)
Carry genes to transfer the own plasmid to another cell
R plasmid
Carry antibiotics resistant gene
Degradative plasmid
Carry specific genes for the utilization of unusual metabolites
Cryptic plasmid
No apparent functional coding genes

4. Plasmid Plasmid copy number Plasmid incompatibility
High-copy-number (relaxed)
10~ 100 copies/cell
Low-copy-number (stringent)
1~4 copies /cell
Plasmid incompatibility
Two or more types of plasmids cannot coexist in the same host cell, if they belong to a single incompatibility group.
But plasmids from different incompatibility groups can be maintained together in the same cell.
Host range of plasmid
Narrow-host-range plasmid
Specific replication origin
Broad-host-range plasmid

5. Features for high-quality cloning vector
Origin of replication
Small size
Decrease in transformation efficiency with plasmids larger than 15 kb
Choice of unique restriction endonuclease recognition sites for cloning DNA
Selectable genetic markers

6. Evolution of Cloning Vectors
Natural plasmids, e.g. Col E1, pSC101
pBR322 (Bolivar and Rodriguez, 1977) : combination of natural plasmids, AmpR, TetR markers, 4.3 kb
p: plasmid
BR: initial of an inventor (F. Bolivar & R. Rodriguez)
322: numerical designation (e.g. order of discovery)
pBR322 derivatives : more unique enzyme sites, other markers
pUC vectors : multiple cloning sites (MCS) into lacZ’ encoding
a-peptide of of b-galactosidase

7. Cloning Foreign DNA into a Plasmid Vector
Alkaline phosphatase treatment of plasmid DNA to prevent self ligation

8. Plasmid Cloning Vector pBR322
Much smaller than the natural plasmid (4361 bp)
resistant to shear
efficient transformation
Origin of DNA replication
stringent control
Plasmid replication is coupled to
that of the host cell. (one or at most
a few copies of plasmid per cell)
relaxed control (pBR322)
copy number: 10~200/cell,
several thousands/cell (with chloramphenicol)
Selection marker
ampicillin resistance (Ampr),
tetracycline resistance (Tetr)

9. Plasmid Cloning Vector pBR322
Single restriction site for a number of enzymes
Restriction site within an antibiotic resistance gene can be used to detect the presence of an insert.

10. Transformation and Selection
Introduction of purified DNA into “competent” bacterial cells
Competency
Natural: active uptake of DNA under high cell density or starvation conditions
Gene transfer between bacterial species
Intake of exogenous DNA (source of nutrient)
Induction of competency of E. coli
Treatment with cold CaCl2  heat shock at 42oC for 2 min

11. Transformation Transformation frequency Transformation efficiency
number of transformed cells/ total cells
Transformation efficiency
number of transformed cells/ amount of DNA
Host cells for cloning
RecA-
No recombination between DNA molecules
Deletion of endA1
No endonuclease production

12. Selection of Transformed Cells
Cloning into BamHI site of pBR322
Selection for clones containing insert DNA in the plasmid
Transformation and selection on plated medium containing ampicillin
Selection for tetracyclin-sensitive clones
Cells with recircularized pBR322 DNA are resistant to both ampicillin and tetracycline.
Confirmation of the plasmid

13. pUC19 Plasmid Vector 2686 bp Ampr Multiple unique cloning sites
Origin of replication
lacI: produces a repressor protein
lacZ’: produces LacZ’ protein which combines with LacZα that is encoded by chromosomal DNA to form an active hybrid β–galactosidase.

14. pUC19 Plasmid Vector

15. pUC19 Plasmid Vector IPTG (isopropylthiogalactoside)
an inducer of the lac operon
X-gal (5-bromo-4-cloroindolyl-β–galactoside)
X-gal  blue color (by β–galactosidase)
Plasmid-containing cells can grow on the ampicillin-containing agar plate.
Intact pUC19  blue colony
pUC19-cloned DNA constructs  white colony

16. Shuttle Cloning Vector
has a second origin of replication that enables the plasmid to replicate in the alternative host cell.