Molecular Biotechnology-Lession 2: Recombinant DNA technology docx

 The first publications describing the successful production and intracellular replication of recombinant DNA appeared in 1972 and 1973..  lacZ encodes β-galactosidase galactosidase La

Recombinant DNA technology

(Gene cloning, genetic engineering)

VIETNAM NATIONAL UNIVERSITY AT HO CHI MINH CITY

INTERNATIONAL UNIVERSITY

Tran Ngoc Duc, PhD

2 Prokaryotic and eukaryotic genes

1/6 Introduction

 Glofish is the first transgenic animal approved to be

consumed by

human in the USA

 The insertion of different

constructs of GFP into the fish

genomes to give different green

colors

 The idea for recombinant DNA was first proposed by Peter

Lobban, a graduate student of Prof Dale Kaiser in the

Biochemistry Department at Stanford University Medical School

 The first publications describing the successful production and intracellular replication of recombinant DNA appeared in 1972 and 1973 Stanford University applied for a US patent on

recombinant DNA in 1974, listing the inventors as

Stanley N Cohen and Herbert W Boyer; this patent was

awarded in 1980

 The first licensed drug generated using recombinant DNA

technology was human insulin, developed by Genentech and Licensed by Eli Lilly and Company

(http://en.wikipedia.org/wiki/Recombinant_DNA)

 Applications of recombinant biotechnology

 Recombinant human insulin

 Recombinant human growth hormone

 Recombinant human blood clotting factor III

 Recombinant hepatitis B vaccine

 Golden rice

 Herbicide resistant crop

 Insecticide resistant crop

2/6 Prokaryotic and eukaryotic genes

 Genes are usually found grouped

promoter

(regulatory region)

 lacZ encodes β-galactosidase galactosidase

(LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose

 lacY encodes β-galactosidase galactoside

permease (LacY), a membrane-galactosidase

bound transport protein that pumps lactose into the cell

 lacA encodes β-galactosidase galactoside

transacetylase (LacA), an enzyme that transfers an acetyl group from acetyl-galactosidase CoA to β-galactosidase galactosides

 Intron: noncoding region, 40-galactosidase 10,000bp

 Exon: coding region, 150-galactosidase 500bp

 5’, 3’ UTR = flanking regions

 Eukaryotic

 Eukaryotic genes are more complex than prokaryotic genes

3/6 Vectors

 Vector is a DNA molecule used as vehicle to transfer and amplify a foreign genetic material into another cell

 There are four types of vector:

o Plasmids

o Virus

o Cosmids

o Artificial chromosomes (BAC, YAC)

 Depending the size of a transgene, a proper vector is used

 A typical plasmid

beta-galactosidase lactamase

 Origin of replication, multi-galactosidase cloning site and selectable marker are common to vectors

 Origin of DNA replication, ori

 High (10-galactosidase 100) & low copy

 White blue screening

 LacZ encode β-galactosidase galactosidase

 β-galactosidase galactosidase cleave X-galactosidase gal

( analog of lactose) to form 5-galactosidase bromo-galactosidase 4-galactosidase chloro-galactosidase indoxyl, which then

spontaneously dimerizes and

oxidizes to form a bright blue

insoluble pigment 5,5'-galactosidase dibromo-galactosidase 4,4'-galactosidase dichloro-galactosidase indigo

 Bacteriophage lamda vector:

 Can carry up to 25kbDNA

 1000 time more efficient than a plasmid

 A virus that can infect bacteria

Cloning in Lambda Vectors

1 Make a partial digest of DNA

2 Ligate the DNA to the arms of the vector

3 Package the DNA into phage particles using

premade mixes

4 Screen and then amplify

5 Store the library for future use as a plate lysate

o A combination of a plasmid and a cos site (DNA sequence, 200bp originally from lambda phage, essential for packaging) which help

to transfer a plasmid into a phage head

o Can carry up to 52 kb DNA

o Unlike plasmid, cosmid can also packaged into a capsid since it contains the cos site and so it can

be used for transduction

 Yeast artificial chromosome (YAC):

o To clone large piece of DNA

which can carry up to 2-galactosidase 3

MbDNA

o Constructed from the

telomeric, centromeric, and

replication origin sequences

needed for replication in yeast

cells

 TEL: The telomere which is located at each chromosome

end, protects the linear DNA from degradation by nucleases

 CEN: The centromere which is the attachment site for

mitotic spindle fibers, "pulls" one copy of each duplicated

chromosome into each new daughter cell

 ORI: Replication origin sequences which are specific

DNA sequences that allow the DNA replication machinery to assemble on the DNA and move at the replication forks

4/6 Cloning a gene

http://www.ndsu.edu/pubweb/~mcclean/plsc731/cloning/cloning2.htm

 Cloning a gene

Gene/DNA isolation

Ligation gene/DNA to a vector

Transform ligated vectore into cell/tissue/ organism

Screening the transfered gene

5/6 Transformations

technology

 Making competent cells with ice-galactosidase cold CaCl2

 A mixture of E coli and vector

 Expose to 2 min at 420C

 Frequency: 1 out of 1000

 Efficiency: 107-galactosidase 108 colonies/µg of plasmid DNA

 Heatshock with E coli

 Subject bacteria to high voltage electric field

 Efficiency: 109 colonies/µg of small plasmid (3kb),

106 colonies/µg of large plasmid (136kb)

 Electroporation

 Mediated by Agrobacteria

 Agrobacteria: a gram-galactosidase negative bacteria

 Use horizontal gene transfer and cause tumor (gall) in plant, either in root or trunk

 The tumor is caused by a plasmid called tumor-galactosidase

inducing plasmid (Ti-galactosidase plasmid or Ri-galactosidase plasmid)

 Specifically, the tumor is caused by a

conjugative DNA fragment on the plasmid, which

is called T-galactosidase DNA

 T-galactosidase DNA carries genes for auxin, cytokinin, nopaline

 Altering balance of plant hormone => cell

division is not controlled

6/6 Detection of cloned

genes/products

 By PCR or southern blotting

 RT-galactosidase PCR or northern blotting

 Protein or enzyme activity

Gene expression in biological systems

1 Microbial expression system

2 Plant expression system

3 Animal expression system

• Plant growth promoting bacteria

 Microbial expression system

 Bacteria: E coli, Bacillus

 Fungi: Yeast, Saccharomyces

2 Plant expression system

 Insect, pathogen, herbicide resistant

 Flower pigmentation

 Nutritional content: Protein, lipid, vitamins, starch

 As bioreactor: Antibodies, polymers, foreign proteins

 Edible vaccines

 Stress and senescence tolerant plants

7 Rapeseed Oil (Canola)

8 Creeping bentgrass (for animal feed)

9 Rice (Golden Rice)

Examples of plant tranformation

using T-galactosidase DNA and Agrobacteria

3 Animal expression system

 DNA microinjection

 Engineered embryonic stem cell

 Cre-galactosidase loxP recombination

 Retrovial vector

 Transgenic mice, goat, sheep, bird, pig, chicken, fish

MicroinjectionCloned gene is injected into nucleus of a fertized egg

Implant fertilized egg into a receptive female

Offspring carry cloned gene

Breeding offspring to establish new genetic lines

 Using glass micropipette to transfer substance into a living cell

 The needle about 0.5-galactosidase 5µm

Embryonic stem cells

 Embryonic stem cell/ Pluripotent cells from

an early stage embryo called blastocyte (4-galactosidase 5 days after fertilization

 Two main characteristics: pluripotent and replicate/make copies indefinitely

 Differentiated into organs/parts of the bofy