isaiah 40:11

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©1999 Timothy G. Standish Isaiah 40:11 11 He shall feed his flock like a shepherd: he shall gather the lambs with his arm, and carry them in his bosom, and shall gently lead those that are with young.

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Isaiah 40:11 11 He shall feed his flock like a shepherd: he shall gather the lambs with his arm, and carry them in his bosom, and shall gently lead those that are with young. Vectors. Timothy G. Standish, Ph. D. + Control. - Control. - Control. Experimental. - PowerPoint PPT Presentation

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Page 1: Isaiah 40:11

©1999 Timothy G. Standish

Isaiah 40:11

11 He shall feed his flock like a shepherd: he shall gather the lambs with his arm, and carry them in his bosom, and shall gently lead those that are with young.

Page 2: Isaiah 40:11

©1999 Timothy G. Standish

VectorsVectors

Timothy G. Standish, Ph. D.

Page 3: Isaiah 40:11

©1999 Timothy G. Standish

Experimental

Transformation Of BacteriaTransformation Of BacteriaThe Griffith ExperimentThe Griffith Experiment

- Control

+ Control

- Control

OUCH!

Page 4: Isaiah 40:11

©1999 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.

Page 5: Isaiah 40:11

©1999 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

Page 6: Isaiah 40:11

©1999 Timothy G. Standish

pUC 18 SequencepUC 18 Sequence

tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaaagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc

Page 7: Isaiah 40:11

©1999 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

Page 8: Isaiah 40:11

©1999 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

Page 9: Isaiah 40:11

©1999 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

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©1999 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

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©1999 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

Page 12: Isaiah 40:11

©1999 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

Page 13: Isaiah 40:11

©1999 Timothy G. Standish

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

Blue colonies - Express -galatosidase which metabolizes colorles 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 b-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

Page 14: Isaiah 40:11

©1999 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

Page 15: Isaiah 40:11

©1999 Timothy G. Standish

A LibraryA Library

The clone of interest

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©1999 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

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©1999 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

Page 18: Isaiah 40:11

©1999 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

Page 19: Isaiah 40:11

©1999 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

Page 20: Isaiah 40:11

©1999 Timothy G. Standish