Recombinant DNA Technology

Screening by Functional Complementation

• Requires strain unable to produce desired product/function

• Cloned DNAs must be in expression vector or include elements required for expression

• Select for restoration of lost function

cDNA Synthesis

• Oligo(dT) primer

• Reverse transcriptase

• Klenow/DNAP I

• RNase H

§ Degrades RNA of DNA:RNA hybrid

• S1 nuclease

§ Degrades ss nucleic acids (unpaired loop)

Enriching for Full Length cDNAs (1)

• Primer has adapter (RE cutting sequence)

• Ribose ends of mRNA are biotinylated

• RNase I degrades ss RNA

• Only full length cDNA is still attached to a biotinylated mRNA (biotin still on 5’end)

• Capture full length copies

Enriching for Full Length cDNAs (2)

• RNase H degrades mRNA

• Add poly(G) to cDNA

• Primer/Adapter with oligo (C)

• DNAP I (Klenow)

• Restriction endonucleases

• Cut Vector

• DNA Ligase

• Transform

Inert Capacities Common Vector Systems

Bacteriophage Lambda Life Cycle

• Lysogenic phage

§ Lysogeny vs. lytic cycle

• Chromosome about 50 kb
• Protein coat for efficient delivery into cells (E. coli)

• Packages DNA 38-52 kb with cos sites at each end

DNA Replication is by rolling circle mechanism

Bacteriophage Lambda Life Cycle

• Lysogenic phage

§ Lysogeny vs. lytic cycle

• Chromosome about 50 kb
• Protein coat for efficient delivery into cells (E. coli)

• Packages DNA 38-52 kb with cos sites at each end

DNA Replication is by rolling circle mechanism

Packaging of l Chromosomes

• Natural DNA is concatemer with cos sites separated by about 50 kb (from rolling circle replication

• DNA is cleaved at cos and inserted into capsid

Mature l Phage

• DNA packaged in protein coat

• Looks much like a lunar lander (actually has six tail fibers)

Phage l cloning vector

• Internal segment deleted (now requires helper phage to replicate)

• Has cos sites intact

• Target DNA inserted between the two l “arms” (up to about 20 kb)

• Recombinant phage infect E. coli cells

High-Capacity Bacterial Vector Systems

§ P1 bacterial systems

§ F plasmid systems

§ BACs (bacterial artificial chromosomes)

Genetic Transformation of Prokaryotes

§ Usually involves CaCl2 and heat shock

§ Transformation frequency about 1/1000

• Electroporation

§ 10-100 times efficient that chemical approach

§ Much better large plasmids (100+ kb)

Electroporation

• Cells suspended in DNA solution in cuvette between two electrodes

• High voltage electric field pulses administered

Conjugation

• Natural system transmitting plasmids from one cell/strain to another

• transferred plasmid and certain proteins which can be provided in trans

• Plasmids of >106 bp can be transferred in this manner

• Can be interspecies

• Tripartite mating and multiple selection

Tripartite Mating

• P. putida difficult to transform

• Transform mobilizable recombinant plasmid into E.coli

• Make culture with P. putida (wt), recombinant E. coli (auxotroph) and E. coli (aux) with conjugative mobilizable plasmid

• Recombinant plasmid transferred - P. putida