©2000 Timothy G. Standish

Genetic Genetic EngineeringEngineering

Timothy G. Standish, Ph. D.

©2000 Timothy G. Standish

Genetic EngineeringGenetic Engineering Genetic engineering involves taking fragments of

DNA and manipulating them using enzymes and in other ways to make new genetic constructs

The “recombinant” DNA made during genetic engineering can be inserted into organisms to change their genetic make-up

In the transformation experiment you have been doing, you have inserted a recombinant piece of DNA called the pBLU plasmid into bacteria. On that pBLU plasmid is the lacZ gene and a gene for antibiotic resistance, both of which the bacteria lacked before you put them into it

©2000 Timothy G. Standish

VectorsVectors If a fragment of DNA is ligated into an appropriate

vector, it can be inserted into cells which will then make many copies of it

Vectors are typically plasmids or viruses that have been engineered to both accept DNA insertions and reproduce inside cells

Cloning is the process of inserting DNA encoding a gene of interest into a vector, then establishing it as a stable part of a cell line.

©2000 Timothy G. Standish

2,686 bp

pUC 18pUC 18A Typical PlasmidA Typical Plasmid

Lac ZGene

Multiple CloningSite

aagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcHindIII SphI PstI SalI XbaI BamHI XmaI KpnI SstI EcoRI AccI SmaI BanII HincII BspMI

Originof Replication

Ampr

Gene

©2000 Timothy G. Standish

pUC 18 SequencepUC 18 Sequence

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaaagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc

©2000 Timothy G. Standish

G

CTTAA

AATTC

G

1 Digestion

2 Annealing of sticky ends

3 Ligation

Ligase

G

CTTAA

AATTC

G

EcoRIEcoRI

R. E.s and DNA Ligase R. E.s and DNA Ligase Can be used to make recombinant DNACan be used to make recombinant DNA

GAATTC

CTTAAG

GAATTC

CTTAAG

G

CTTAA

AATTC

G

4 Recombinant DNA

©2000 Timothy G. Standish

Host Cell

Cloning Into pUC18Cloning Into pUC18

pUC18

LacZ

Ampr

R. E.Digestion

Addition of ligase joins nicks and makes a single recombinant plasmind

R. E.Digestion

Matching sticky ends anneal

Transformationof cells with the recombinant plasmid

©2000 Timothy G. Standish

So How Do You Know IfSo How Do You Know IfYou Cloned Something?You Cloned Something?

IPTG - Induces expression of lacZ

X-Gal - A lactose analog which turns blue when split by -galactosidase

Ampicillin - Kills all bacteria that lack the plasmid

©2000 Timothy G. Standish

X-GalX-Gal5-Bromo-4-chloro-3-indolyl 5-Bromo-4-chloro-3-indolyl --DD-galactopyranoside-galactopyranoside

OH

O

OH

HOCH2

HO

GlucoseO

O

OH

HOCH2

HO

HO Galactose

LactoseO--D-galactopyranosyl-(1->4)--D-glucopyranose

©2000 Timothy G. Standish

-GalactosideaseLac Z

gene product

X-GalX-Gal5-Bromo-4-chloro-3-indolyl 5-Bromo-4-chloro-3-indolyl --DD-galactopyranoside-galactopyranoside

NH

Br

Cl

O

O

OH

HOCH2

HO

HO Galactose

X-Gal(Colorless)

H2O

©2000 Timothy G. Standish

-Galactosidease

X-GalX-Gal5-Bromo-4-chloro-3-indolyl 5-Bromo-4-chloro-3-indolyl --DD-galactopyranoside-galactopyranoside

OH

O

OH

HOCH2

HO

HO Galactose

Blue

NH

Br

Cl

HO

©2000 Timothy G. Standish

So How Do You Know IfSo How Do You Know IfYou Cloned Something?You Cloned Something?

Blue colonies - Express -galactosidase which metabolizes colorless X-gal to blue and turn blue thus lacZ is not disrupted and there is no foreign DNA cloned

Cloned fragments disrupt lacZ thus make no -galactosidase and colonies remain white

IPTG - Induces expression of lacZ

X-Gal - A lactose analog which turns blue when split by -galactosidase

Ampicillin - Kills all bacteria that lack the plasmid

©2000 Timothy G. Standish

LibrariesLibraries If all the DNA from an organism is digested with a restriction enzyme

and cloned into a plasmid, many different recombinant plasmids will be made, each with a different fragment of DNA cloned into it

Once inserted into host cells or viruses, this collection of many different recombinant plasmids is called a “library”

When the whole genome of an organism is used as the starting point for cloning, it is called a “shotgun clone”

A library constructed using shotgun cloning may contain hundreds of thousands of different recombinant plasmids

Screening is the process of sifting through the library to find the clone of interest

©2000 Timothy G. Standish

A LibraryA Library

The clone of interest

©2000 Timothy G. Standish

Library ScreeningLibrary Screening Libraries tend to have a lot of clones, only one of which has the

sequence of interest Screening a library is the process of eliminating those clones that do

not contain the sequence of interest and locating the clone that does There two major techniques are used for screening: Hybridization screening - In which DNA from a library is bound to a

membrane, then the membrane is exposed to a probe that should base pair (hybridize) to the sequence of interest

Expression vectors may be used so that if the gene for a protein is cloned, the protein is made. To do this, you must be able to detect the protein

©2000 Timothy G. Standish

cDNA LibrariescDNA Libraries Because of the large size of libraries and the tedium of screening,

anything that can be done to limit library size is a good thing Protein coding regions of most eukaryotic genomes make up only

a small percentage of the total DNA (3% in humans) Most cells only express a small subset of an organism’s genes By using reverse transcriptase, a cDNA copies of the mRNA

being produced in a group of cells can be made Cloning cDNA to make a library produces a much smaller library

enriched with the part of an organism’s genome that is of most interest

©2000 Timothy G. Standish

Rev.Trans.

TTTTTTTTTTTT5’5’

cDNA Library ConstructioncDNA Library Construction

TTTTTTTTTTTT5’

TTTTTTTTTTTT5’5’

cDNA after RNase treatment

AAAAAAAAAAA3’5’

mRNA

AAAAAAAAAAA3’5’

mRNAcDNAhybrid

Insert into vector

AAAAAAAAAAA3’5’

Reverse transcription

TTTTTTTTTTTT5’5’

Double-stranded cDNA after DNA polymerase

RNase

A

A

AA AA

A

AAAAAAAAAAA3’5’DNAPol

©2000 Timothy G. Standish

©2000 Timothy G. Standish

An Expression VectorAn Expression Vector

II

III

AatII

pPROTet.E

SacIt0

Myc tagEK siteMCS

ColE1

Cmr

XbaI

T1

• pPROTet.E is a commercially available plasmid sold by Clontech

• It is specifically designed to allow efficient control of expression