Genetic Engineering

Biotechnology

YUSRON SUGIARTO

The manipulation of a trait in an organism to create a desired

change

What is Genetic Engineering?

We have been manipulating DNA for generations! Artificial breeding

creating new breeds of animals & new crop plants to improve our food

Animal breeding

Breeding food plants “Descendants” of the wild mustard

the “Cabbage family”

Breeding food plants

Evolution of modern corn (right) from ancestral teosinte (left).

A Brave New World

The code is universal Since all living

organisms… use the same DNA use the same code

book read their genes

the same way

TACGCACATTTACGTACGCGGATGCCGCGACT

ATGATCACATAGACATGCTGTCAGCTCTAGTAG

ACTAGCTGACTCGACTAGCATGATCGATCAGC

TACATGCTAGCACACYCGTACATCGATCCTGA

CATCGACCTGCTCGTACATGCTACTAGCTACTG

ACTCATGATCCAGATCACTGAAACCCTAGATC

GGGTACCTATTACAGTACGATCATCCGATCAGA

TCATGCTAGTACATCGATCGATACTGCTACTGA

TCTAGCTCAATCAAACTCTTTTTGCATCATGAT

ACTAGACTAGCTGACTGATCATGACTCTGATCC

CGTAGATCGGGTACCTATTACAGTACGATCATC

CGATCAGATCATGCTAGTACATCGATCGATACT

GCTACTGATCTAGCTCAATCAAACTCTTTTTGC

ATCATGATACTAGACTAGCTGACTGATCATGAC

TCTGATCCCGTAGATCGGGTACCTATTACAGTA

CGATCATCCGATCAGATCATGCTAGTACATCGA

TCGATACT

human genome

3.2 billion bases

Can we mix genes from one creature to another?

YES!

Green Fluorosceint Protein (GFP)

Uses of genetic engineering Genetically modified organisms (GMO)

enabling plants to produce new proteins ___________________________: BT corn

corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn)

___________________________: fishberries strawberries with an anti-freezing gene from

flounder

___________________________: golden rice rice producing vitamin A

improves nutritional value

Basic steps in genetic engineering

1. Isolate the gene 2. Insert it in a host using a vector 3. Produce as many copies of the host as

possible 4. Separate and purify the product of

the gene

Gene Cloning Techniques

1- Grow the target

microorganism

2.Extract/isolate

DNA

DNA target

3- Digest

fragment DNA

with

restriction

enzymes

4- Insert DNA

fragments in a

plasmid cloning

vector

Recombinant

Each bacteria will grow to

form an individual colony

Continued

“Vibrio DNA

library”

5-

Transform

E. coli with

library

Each bacteria will receive

a single plasmid from the

library

Tools

1. DNA you want to clone

2. Restriction endonucleases (molecular scissors)

3. Cloning vector (e.g. pGEM, pBR322…)

4. Ligase enzyme (molecular glue)

Step 1: Isolating the gene

Step 1: Isolating the gene

Cutting DNA DNA “scissors”

used by bacteria to cut up DNA of attacking viruses

EcoRI, HindIII, BamHI

cut DNA at specific sites enzymes look for specific base sequences

GTAACGAATTCACGCTT

CATTGCTTAAGTGCGAA

GTAACG|AATTCACGCTT

CATTGCTTAA|GTGCGAA

Restriction enzymes Cut DNA at specific sites

____________________________

GTAACG AATTCACGCTT

CATTGCTTAA GTGCGAA

GTAACGAATTCACGC

TT

CATTGCTTAAGTGCG

AA

restriction enzyme cut site

restriction enzyme cut site

Sticky ends Cut other DNA with same enzymes

leave “sticky ends” on both can glue DNA together at “sticky ends”

GTAACG AATTCACGCTT

CATTGCTTAA GTGCGAA gene

you want

GGACCTG AATTCCGGATA

CCTGGACTTAA GGCCTAT

chromosome want to add

gene to

GGACCTG AATTCACGCTT

CCTGGACTTAA GTGCGAA combined

DNA

Restriction Endonucleases

• Restriction endonucleases, a.k.a. “restriction enzymes” or “enzymes” by

molecular biologists.

• Type II restriction enzymes recognize and cut specific DNA sequences

5’-NNNAAGCTTNNN-3’

3’-NNNTTCGAANNN-5’

Example

• Hind III (Haemophilus influenza Rd)

– Recognizes: AAGCTT

– Cuts in between the two A’s

AAGCTT A AGCTT

TTCGAA TTCGA A

Types of Sticky Ends

5’ overhangs (HindIII)

5’AAGCTT 3’ 5’A 5’ AGCTT3’

3’TTCGAA 5’ 3’TTCGA 5’ A 5’

3’ overhangs (KpnI)

5’ GGTACC 3’ 5’ GGTAC 3’ C 3’

3’ CCATGG 5’ 3’ C 3’ CATGG 5’

Types of Overhangs

Sticky ends Examples include HindIII & KpnI

Blunt Ends Example SmaI Recognize CCCGGG Cut between C and G CCCGGG CCC GGG GGGCCC GGG CCC

Sticky ends help glue genes together TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCA

CGCTT AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGT

GCGAA

gene you want cut sites cut sites

AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTT

TTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA

chromosome want to add gene to cut sites

AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAA isolated gene

sticky ends

chromosome with new gene added TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC

CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC

sticky ends stick together

DNA ligase joins the strands ________________ DNA molecule

Why mix genes together?

TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC

CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC

Gene produces protein in different organism or different individual

aa aa aa aa aa aa aa aa aa aa

“new” protein from organism ex: human insulin from bacteria

human insulin gene in bacteria

bacteria human insulin

How can bacteria read human DNA?

Step 2: Inserting gene into vector

Vector – molecule of DNA which is used to carry a foreign gene into a host cell

Plasmid Vector: pBR322

First modern cloning vector (1976)

pBR322

• Contains: 1. colE1 origin of replication (ORI)

Bacteria plus

plasmid

Non-transformed

bacteria

Nutrient media

plus antibiotic

Overnight

growth

Only colonies

from bacteria that

have plasmid

pBR322

• Contains:

2. Selectable Markers:

• Ampicillin Resistance

(β-lactamase gene)

• and Tetracycline

Resistance (tet gene)

pBR322

• Contains: 3. A few good restriction sites for inserting

foreign DNA

PstI

Eco

RI Bam

HI

BamH1

BamH1

Your favorite

DNA Digest

with

BamH

1

and

ligate

PstI

Eco

RI Bam

HI Bam

HI

Your

favorite

DNA

pBR322

• Nice Features: √ 200 copies per E. coli cell √ Makes double stranded DNA √ All modern cloning vectors are based on

pBR322

• Advantages over pBR322

1. Makes 1000’s of copies/cell

2. Small size at 2.7 kilobase pairs (kb) = easier

uptake by E. coli

Next Generation: pUC Plasmids

Step 3: inserting vector into host

Bacteria Bacteria are great!

one-celled organisms reproduce by mitosis

easy to grow, fast to grow generation every ~20 minutes

A way to get genes into bacteria easily insert new gene into plasmid insert plasmid into bacteria bacteria now expresses new gene

bacteria make new protein

+

transformed

bacteria gene from

other organism

plasmid

cut DNA

recombinant

plasmid

vector

glue DNA

Blue/White Selection Bacteria plus empty

plasmid Non-transformed bacteria

Only colonies

from bacteria that

have plasmid

Nutrient media plus

antibiotic plus X-Gal

Overnight growth

Bacteria with

plasmid plus insert

Colonies with insert - white

Colonies w/o insert - blue

Grow bacteria…make more

grow

bacteria

harvest (purify)

protein

transformed

bacteria

plasmid

gene from

other organism

+

recombinant

plasmid

vector

Applications of biotechnology

any Questions?