CLONING VECTORS FOR E.COLI

BASED ON E.COLI PLASMIDS

Different types

Principle

Nguyen Thu Giang-IPMB, VUB

#

General

#

What is a plasmid?

• Plasmids are small, extrachromosomal pieces of bacterial DNA that are often antibiotic resistant

• They are „shuttle vectors‟ to create, produce,

and maintain recombinant DNA

• An example of the first cloning vector for

E.coli based on E.coli plasmids is pBR322

And pUC18 now is the most commonly used

#

Characters

• A way to maintain in engineered DNA in

the cell’s progeny

• A way to identify (or select) the cells

containing DNA

• A place to insert the gene or piece of

DNA you want to work with

#

General properties of plasmid

vectors:

• small, easily isolated, ease of transformation

• high copy number

• polylinkers or multiple cloning sites (MCS)

• selection for transformation (drug resistance)

• ease of screening (lacZ)

#

Specific properties of plasmid

vectors:

• they must to appropriate to the host, e.g. ori, selection

• they must be appropriate to your objectives

- expression or non-expression

- expression of RNA or protein

- integration and non-integration

- low or high copy number

- ease of sequencing

- facilitate library construction (“the size problem”)

- work in different species (shuttle vectors)

#

What is a plasmid?

#

#

Different types

#

pBR322

• Nomenclature

– ‘p’ indicates that it is indeed a plasmid

– ‘BR’ identified the laboratory in which the

vector was originally constructed

– ‘322’ distinguishes this plasmid from others

developed in the same laboratory

#

pBR322

• pBR322 is an example of an early

cloning vector that replicates in E.coli.

• pBR322 has some significant features:

– It has a small size of 4363 bp

– It carries two sets of antibiotic resistance

genes

– It has reasonably high copy number

#

pBR322• Ori allows independent

replication

• Single cleavage sites for various restriction enzyme (BamHI ect)

• tet resistance gene

• amp resistance gene

• Mechanism:• insertion of foreign DNA

at BamHI site

• tet resistance gene inactivated

• transformants carrying foreign DNA are amp resistant but tetracycline sensitive

#

pBR322

• The pedigree

– amp gene

originally resided

on the plasmid

R1

– tet gene is

derived from R6-5

– The replication

origin of pBR322

is originally from

pMB1

#

“Evolution” of Plasmid Vectors

pUC series

pGEM series

Protein Purification

pGEX series

Regulated Expression

pET, pBAD, pTET

CosmidsPhagmid

λZAP

#

pBR327

• pBR327, one of the first derived from

pBR322, was produced by removing

1089 bp from pBR322:

– pBR327 has a higher copy number

– pBR327 is non-conjugative

Both are now no longer widely used

#

pUC series

• next generation, although only replication origin and amp genes remain

• used in BIO lab• collects all restrictions sites into MCS or polylinker

• 3 advantages:– fortuitous: the manipulations involved in construction

of pUC series were accompanied by a chance mutation

– Identification of recombinant cells can be achieved by a single step process and pUC series introduces screening with x-gal

– The clustering of restriction sites allows DNA fragments with two sticky ends.

#

#

pGEM3Z

• next generation,

2750bp

• very similar to pUC

vectors but has two

promoters

• introduces RNA

expression for in vitro

or in vivo expression.

#

Principle

#

CLONING PROCESS

• Gene of interest is cut out with RE

• Host plasmid is cut with same RE

• Gene is inserted into plasmid and ligated with ligase

• New plasmid inserted into bacterium (transform)

#

PLASMID CLONING

STRATEGY

• Involves five steps:

1.Enzyme restriction digest of DNA sample.

2.Enzyme restriction digest of DNA plasmid vector.

3.Ligation of DNA sample products and plasmid vector.

4.Transformation with the ligation products.

5.Growth on agar plates with selection for antibiotic resistance.

#

STEP 1. RE DIGESTION OF DNA

SAMPLE

#

STEP 2. RE DIGESTION OF

PLASMID DNA

#

STEP 3. LIGATION OF DNA

SAMPLE AND PLASMID DNA

#

STEP 4. TRANSFORMATION OF

LIGATION PRODUCTS

• The process of transferring exogenous DNA into

cells is call “transformation”

• There are basically two general methods for

transforming bacteria. The first is a chemical

method utilizing CaCl2 and heat shock to

promote DNA entry into cells.

• A second method is called electroporation based

on a short pulse of electric charge to facilitate

DNA uptake.

#

CHEMICAL TRANSFORMATION

WITH CALCIUM CHLORIDE

#

TRANSFORMATION BY

ELECTROPORATION

#

STEP 5. GROWTH ON AGAR

PLATES

#

STEP 5

• Blue colonies represent Ampicillin-resistant bacteria

that contain pVector and express a functional alpha

fragment from an intact LacZ alpha coding

sequence.

White colonies represent Ampicillin-resistant

bacteria that contain pInsert and do not produce

LacZ alpha fragment

#

Thanks for your attention!

#

Merry Christmas and Happy

New Year