Chapter 4 Recombinant DNA Technology

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

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

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

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

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

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

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)

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.

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

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

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

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.

pUC19 Plasmid Vector

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

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