1

MCB 3020, Spring 2005

Chapter 31:Genetic Engineering and

Biotechnology I

2Genetic Engineering II. Genetic EngineeringII. Cloning vectors

3I. Genetic Engineering

i. Some usesii. Restriction enzymesiii. DNA cloningiv. DNA library

4Genetic EngineeringDNA manipulation using molecular biology techniques

• DNA cloning• identification of genes of interest• expression of genes to make a

desired product

Typical procedures

5i. Some uses of genetic engineering• Industrial or biotechnology products

– (eg. alkaliphilic proteases, Taq polymerase) • Medical products

– (eg. insulin, hepatitis B vaccines, gene therapy)

• Agriculture and Environment – (eg. plant resistant to pesticides, insects, disease)

• Basic research – (answers to fundamental questions about life)

6ii. Restriction enzymes

a. natural roleb. recognition sequencec. cut sitesd. modification enzymes

7ii. Restriction enzymes

Enzymes that break double-stranded DNA at specific sequences.

Used to protect bacteria fromviruses (by cutting viral DNA)Bacterial DNA is protected bymodification enzymes. TB

a. Natural role

8b. Recognition sequence DNA sequence where cutting occurs

EcoRI recognition sequence

GAATTCCTTAAG

cut sites

Palindromic recognition sequence TB

9

G AATTCCTTAA G

After cutting

double stranded break

sticky ends

TB

10

TTTAAAAAATTT

TTTAAA

AAATTT

After cutting: blunt ends

DraI

DraI recognition sequence

TB

11c. Modification enzymes• covalently modify DNA, often by methylation

• protect bacteria from their own restriction enzymes

• recognize the same site on DNA as the corresponding restriction enzyme

• prevent cutting by the corresponding restriction enzyme

12

GAATTC

CH3

EcoRI methylaseTypical modification enzyme

CTTAAG

methylationCH3

TB

After modification, the EcoRI restriction enzyme will NOT recognize the methylated DNA.

13iii.DNA cloningIsolation and insertion of a DNA fragment (insert) into a vector.

ori

+

Cloning vectora small independently replicating genetic element into which genes can be recombined

14Basic steps of DNA cloning

2. Digest source DNA and vector using restriction enzymes3. Ligate the source DNA to the vector4. Introduce DNA into a host5. Identify the clone of interest

TB

1. Isolate source DNA

15source DNA cloning vector

One to many clones representing the source DNA

TB

2. Digest DNA and vector

cloned DNA(insert) vector

3. Ligate

host cell4. Introduce DNA into host

5. Identify clone of interest

1. Isolate

161. Isolate source DNAx x

eg. BamHI

xsource DNA

2. Digest source DNA and vector using restriction enzymes

gene of interest

17

restriction site

3. Ligate source DNA into vectors.

source DNA

ori

cloning vector

+

x+

cloned DNA

(insert)

X

184. Introduce DNA into a host.

+E. coli

DNA library

Plate on agar

X

X

195. Identify the clone of interestPlate on selective medium to find colonies with cloned DNA

E. coli with cloned DNA

Identify colonies with gene of interest

20iv. DNA libraryA large number of clones representingthe entire genome of an organism.

The source DNA for DNA libraries istypically the genomic DNA.

Introduce into host cells; plate

X

x

21II. Cloning vectors

A. important featuresB. examples

restriction site

ori

ApR

22

1. means of replication (ori) 2. unique restriction sites (single cut)3. selectable markers 4. gene inactivation marker

A. Important features

restriction site

ori

ApR

ApR = ampicillin resistance gene

TcR

TcR = tetracycline resistance gene

23Selectable markerIn genetic engineering, a gene whose product can be used to select the cells that carry the plasmid of interest

Gene inactivation markerIn genetic engineering, a gene that is disrupted (inactivated) when a second gene of interest is cloned into the plasmid or DNA

244a. Selectable markers and gene inactivation

Uncut vector allows cells to grow on ampicillin (Ap) and tetracycline (Tc).

What happens when foreign DNA is inserted into the BamH1 site?

BamH1

ori

ApR

TcR

transform E. coli

+ ampicillinreplica plating + ampicillin

+ tetracycline

• the TcR (tetracyline resistance) gene is inactivated

25Selectable markers and gene inactivation

When foreign DNA is inserted, • TcR gene is inactivated • cells will grow on Ap, but NOT tetracycline

BamH1

ori

ApR

TcR

BamH1digest

Inactivated TcR

ApR

TcRApR

+

TcRApR

insert

26

Cells containing the cloned DNA (insert), are Ap-resistant (ApR) but Tc-sensitive (TcS ).

replica plating + ampicillin

+ tetracycline

In this gene inactivation system, what happens when E. coli is transformed with a mixture of vector and [vector with insert]?

+ ampicillin

274b. Another gene inactivation marker is the lacZ gene (codes for beta-galactosidase)

BamH1

ori

ApR

lacZ

ClBr

N

OO

X-gal(CLEAR)

BLUE product

beta-galactosidase cleaves X-gal and produces a blue color

ClBr

N

HO

OOH

beta-galactosidase

28Colonies containing vector WITHOUT an insert are blue.

+ ampicillin+ X-gal

(We don't want these.)

BamH1

ori

ApR

lacZ

29When foreign DNA is inserted, it inactivates lacZ

• beta-galactosidase is not made• X-gal is not cleaved • colonies with insert are white, NOT blue

XX-gal(CLEAR)

+ ampicillin+ X-gal

X (LacZ-)insert

30B. Examples of cloning vectors

1. Plasmids2. Phage3. Cosmids4. YACs

31

pBR322

BamHI

BamHI = unique restriction site

ApR

ApR = ampicillin resistance gene

TcR

TcR = tetracycline resistance gene

ori

ori = origin of DNA replication

vector1. plasmid vector (holds ~10 kb)

source DNABamHI sites

32BamHI digestion

mixture of [vector with cloned DNA], and vector

ligation

clonedDNA

TcR source DNA

TB

33

a. Phage lambda ()

dsDNA

1/3 of genome non-essential for lytic growth

2. Phage vector (holds about 20 kb)

(can replace this section with foreign DNA)

TB

34e.g. of phage vector: Charon 4A (genetically altered derivative)

lacZ gene cos site

EcoRI sites

1. restriction2. ligation

cloned DNA TB

353. Package the cloned DNA into capsids in vitro.

4. Infect host cells and plate to obtain plaques

lawn of E. coli cellsplaques (regions of dead cells caused by lytic phage)

36

blue plaques (LacZ+)

clear plaques (LacZ-)

5. Isolate DNA from clear plaques. (Blue plaques do NOT have insert. We don't want these.) TB

37b. Phage M13 vectors

• Phage M13: a ssDNA virus that has a dsDNA replicative form

• Used to produce ssDNA for DNA sequencing and site-directed mutagenesis

• Double-stranded replicative form is used for cloning

383. Cosmid (holds up to 45 kb)

Plasmids with cos (cohesive end) sitesfor in vitro packaging into capsids.

plasmid

cos

1. clone DNA fragments2. linearize3. package in vitro

394. Yeast Artificial chromosomes (YACs) (holds up to 800 kb)

oritelomerescentromerecloning siteselectable marker200-800 kb inserts

(Human genome ~ 3 x 109 bp or 3 x 106 kb)

Features of YACS:

40Comparison of clone sizes

Plasmids up to ~10 kbCharon phage up to ~ 20 kbCosmids up to ~ 45 kbYACS up to ~800 kb

41C. Hosts for cloning vectors

Escherichia coliBacillus subtilisSaccharomyces cerevisiae (yeast)mammalian cells

42Study objectives1. Name three procedures typically used in genetic engineering.2. What are some uses of genetic engineering? Know the examples presented.3. What are restriction and modification enzymes? What is their natural role? Describe the general features of the recognition site of restriction enzymes. You do NOT need to memorize the sequences of the recognition sites.4. What is DNA cloning? What is a cloning vector? 5. Understand in detail the basic steps involved in cloning DNA.6. What is a DNA library? What is the typical source DNA for a library?7. Know the important features of a cloning vector and their roles in cloning.8. Describe how antibiotic resistance genes and the beta-galactosidase gene can be used to determine if foreign DNA has been inserted into a vector.9. Understand why the following are important for cloning vectors: selectable markers, gene inactivation, means of replication, unique restriction sites.10. How the following are used in DNA cloning: plasmid vectors (example, pBR322) phage vectors (examples, Charon 4A and M13) cosmids, and YACs. 11. Compare and contrast the different DNA cloning vectors. What features are specific to each cloning vector? 12. Know that specific host cells facilitate cloning. Know the examples presented.

43

MCB 3020 Spring 2005

Chapter 31:Genetic Engineering and

Biotechnology II

44Last time:I. Genetic EngineeringII. Cloning vectors

III. Identifying clones of interestIV. Expression vectorsV. Polymerase chain reaction (PCR)VI. Cloning and expression of mammalian genes in bacteriaVII. Applications of genetic engineering

Today:

45III. Identifying clones of interest

A. antibodiesB. DNA and RNA probesC. complementation

46A. antibodies (immunoglobulins)soluble immune system proteins that bind specific antigens*

TB(*Antigens are "nonself" (foreign) molecules that interact with

components of the immune system.)

This antigen is a protein.

47Using antibodies to identify clones

3. If a DNA clone expresses protein X, it includes the gene for protein X

1. Purify protein of interest (protein X).X

Y2. Prepare antibody ( ) that specifically

binds to protein X.Y

4. Use antibody to test clones for production of protein X.

TB

48

transformantcolonies

DNA Librarytransform E. coli

1. replica plate cells to filter paper

transformant cells on filter paperTB

Using antibodies to identify clones of interest

492. lyse cells

3. bind the antibody4. detect the antibody

contains a DNA cloneexpressing the protein of interest

TB

50B. DNA and RNA probes

Probe: labeled DNA or RNA that can bind a particular DNA by complementary base paring.

(Probes can be short single-stranded oligonucleotides with a radioactive or fluorescent label attached)

32P

51Uses of DNA probes

1. Detect DNA with a sequence related to a DNA of known sequence.2. Detect genes that encode proteins of partially known sequence.

TB

52

1. lyse cells

3. bind and detect probe2. denature DNA

contains a clone with sequences complementary to the probe

transformant cells on filter paper

TB

53C. Complementation: How could genes of interest be identified by complementation?

Restoration of the wildtype phenotype by a second DNA moleculeTB

X

human DNA library

mutation

X

E. coli coenzyme B12 mutant(can't make coenzyme B12)

X

54IV. Expression vectors

A. Factors affecting protein expressionB. Typical expression vector

gene forregulatoryprotein

P O

ApR

Vectors used to produce large amounts of protein.

ori

55Expression vectors

Vectors used for the production ofproteins.

usually used to get a high level of gene expression

TB

56A. Factors affecting protein expression

2. Promoter strength and regulation3. Translation initiation4. Codon usage5. Protein and mRNA stability

TB

1. Gene copy number

57B. Typical expression vector

P O

P = promoterO = operator

gene ofinterest

selectablemarker

unique restriction site

ori

lacI gene(encodesrepressorprotein)

TB

58

P O lacZ lacY lacA

Lactose ( ) induces the expression of lac genesor whatever genes follow the lac promoter.

CAPsite

Some repressor proteins mediate gene induction.

+

P O gene of interest

normal lac operon

genetically engineered gene

protein of interest

59V. Polymerase chain reaction (PCR)

A. applicationsB. reaction componentsC. procedure

Process for producing large amounts of DNA from a small amount of template DNA.

60A. PCR applications

gene cloningmutagenesisamplification of related sequences

TB

amplification of small amounts of DNA for

61B. PCR reaction components

the 4 deoxynucleotidesbuffer

template (~104 molecules)thermostable DNA polymerase (Taq or Pfu polymerase)

2 DNA primers (1017 molecules)

TB

62The 2 DNA primers bind on opposite strands of DNA

5'

5'

Primer #2Primer #1

Heat to separate strandsCool to anneal to primers

primers

template

TB

63

1. denature template DNAtemplateprimers

DNApolymerase

denature at 94°C

C. procedure

TB

64

anneal at ~ 50ºC

2 anneal primers

primers bind by complementary base pairing

TB

65

extend at 72ºC

3. extend with DNA polymerase

4. repeat steps 1-3, ~ 35 times (35 cycles)TB

66

denature

second cycle

TB

67

anneal

second cycle

TB

68

extend

second cycle

TB

6935 cycles

template

final product

primers are incorporated into productTB

70

= (number of templates) x 2 (number of cycles)

= (1) x 235 = 3.4 x 1010 molecules

Amount of product from 1 molecule

34,000,000,000

TB

71

A. Problemsintronslarge genomesposttranslational modifications(like glycosylation, attaching a sugar)

One solution to the intron problem:cDNA ("complementary DNA")

VI. Cloning and expression of mammalian genes in bacteria

TB

72B. cDNA

mRNA AAAA...TTTT...

alkali (removes mRNA)

reverse

AAAA...TTTT...

transcriptaseprimer

TB

73

DNA polymerase

clone

specific nuclease

cDNA

TB

74VII. Applications of genetic engineering

A. General usesB. Mammalian proteinsC. VaccinesD. PlantsE. Gene transfer to plants by bacteria

TB

75A. General uses

Microbial fermentations (eg. antibiotic production)

VaccinesMammalian proteins

TB

Transgenic plants and animalsEnvironmental biotechnologyGene therapy

76B. Mammalian proteinsInsulinalpha-interferonclotting factors

TB

C. VaccinesHepatitis B

77D. Genetic engineering in plants

Disease resistanceImproved product qualityProduction of pharmaceuticals

TB

Herbicide resistanceInsect resistance

78

cloned DNA

transfersequences

E. Gene transfer to plants by bacteria

plasmid used for gene transfer

KanR

KanR = kanamycin resistanceTB

79

Plant cell genome

Agrobacteriumtumefaciens

transgenic plantregeneration

D-Ti

Provides genesneeded for DNATransfer

TB

80Study objectives1. Understand the details of how antibodies, nucleic acid probes and complementation are used to identify particular clones.2. Know the main factors that affect protein expression from expression vectors.3. Understand the polymerase chain reaction, its uses, and the details of the procedure presented in class.4. Understand how cDNA is made and how it solves some of the problems of cloning eukaryotic genes.5. What are some of the applications of genetic engineering?6. Understand how Agrobacterium can be used to transfer genes to plants.