Cloning Vectors

Plasmids

small, circular double stranded DNA molecules

components of plasmid cloning vectors:

1. origin of replication (ori)

site where DNA replication is initiated

most common plasmid cloning vectors

– contain ori from plasmid pMB1

pMB1 ori functions in E. coli

– not in other organisms

broad-host-range plasmids

– replicate in > 1 species

e.g. IncQ plasmids

– replicate in many Gram negative bacteria

shuttle vectors

– contain origins of replication for different species

e.g. YEp (yeast episomal plasmid)

– contains: pMB1 ori

S. cerevisiae ARS

(autonomously replicating sequence)

– plasmid replicates in both species

control of plasmid replication

most wild type plasmids

– control number of plasmids in cell

low copy number plasmids – have stringent control

– plasmid replication same rate as genome replication

– 1-2 copies per cell

high copy number plasmids – have relaxed control

– plasmid replication independent of genome

– > 15 copies per cell

pMB1 ori – relaxed

– allows amplification with chloramphenicol

chloramphenicol – antibiotic

– interferes with protein synthesis

– stops genomic DNA replication

(protein synthesis required)

pMB1 replication does not require protein synthesis

– plasmid replication continues

– 100s-1000s plasmids per cell

copy number increased by deleting regulatory genes

– e.g. pUC plasmids – > 500 copies per cell

– pMB1 derivatives lacking regulatory region

higher copy number usually preferred in cloning vectors

– more cloned DNA produced

–problem if cloned gene is expressed & is toxic

plasmid incompatibility

– more than one type of plasmid with similar ori

– cannot be present in same cell

reason – copy number regulatory systems

– plasmids with same ori

– interfere with each other’s replication

copy number for both plasmids kept low

–statistically unlikely both maintained in same cell

2. marker genes for selection and/or screening

selection – killing cells that lack specific gene

– e.g. antibiotic resistance genes

– plasmid with antibiotic resistance genes

– allows cells to survive on media containing antibiotics

only cells containing plasmid form colonies

screening

– testing for difference in phenotype between cells

– depending whether functional copy of gene present

e.g. pBR322 – artificial plasmid (Fig. 2.1)

– constructed from: pMB1 ori

antibiotic resistance genes (transposon, plasmid)

2 antibiotic resistance genes

– ampicillin & tetracycline resistance

if insert cloned into one gene – gene inactivated

other (functional) antibiotic resistance gene

– allows selection for plasmid

colonies containing plasmid

– replica plated to media with antibiotic being screened for

– colonies that do not grow on screening media

– contain plasmid with insertion inactivated gene

– plasmid contains cloned DNA

second example – pUC vectors (Fig. 2.8)

allow screening by insertion inactivation of lacZ gene

LacZ gene product

– cleaves lactose to glucose and galactose

also cleaves X-gal (Fig. 2.6)

(5-bromo-4-chloro-3-indolyl-β-D-galactoside)

X-gal – colourless – cleavage releases blue pigment

lacZ⁺ cells produce blue colonies on media with X-gal

pUC vectors contain lacZ ' gene

– codes for N-terminal domain of LacZ

– referred to as “alpha peptide”

used in host cells containing lacZ ΔM15 (e.g. DH5α strain)

– mutant with part of 5' end of gene deleted

if lacZ ' and lacZ ΔM15 in same cell

– get alpha-complementation

gene products assemble into functional enzyme complex

if only 1 of 2 deleted genes present

– or if lacZ ' is inactivated by cloned insert

– get (off) white colonies

complications:

lacZ gene not expressed constitutively

– X-gal does not activate gene expression

must use IPTG as inducer (Fig. 2.7)

(isopropyl-β-D-thio-galactoside)

small inframe insertions may not inactivate α peptide

– still get blue colonies (often lighter – less activity)

3. Unique restriction endonuclease (RE) sites

– allow inserts to be cloned in specific sites on plasmid

– e.g. within marker genes

important factors in determining number of unique sites

– size of plasmid – small is better

– more DNA present, more chance of RE sites

– depending on random chance

cloning vectors have most excess DNA removed

– e.g. 3' end of lacZ gene

– addition of unique sites to plasmids

– e.g. pUC18 & pUC19 vectors

– contain a multiple cloning site within lacZ '

sequence was synthesized as an oligonucleotide

– then cloned into earlier pUC vectors

(lower numbers)

pUC18 & pUC19

– have multiple cloning site in opposite directions

– allows genes to be cloned in different orientations

– may affect expression of genes

4. transmissability

many wild type plasmids are transmissable by conjugation

requires: tra – codes for pili

mob – gene product nicks DNA at nic/bom site

– starts rolling circle replication

cloning vectors are usually disabled

– by deletion of some/all conjugation functions

prevents recombinant DNA transfer to wild bacterial strains

e.g. pBR322 has no tra or mob regions

– cannot transfer itself

– does have nic & bom sites

– can be transferred if tra & mob present in same cell

– e.g. on “helper plasmid”

pUC vectors lack nic & bom sites

– cannot be transferred, even with tra & mob present

5. Promoters for gene expression

– some plasmids contain controllable promoters

– flanking multiple cloning site

allow expression of cloned genes in E. coli