Set 4: Gene cloning

Where does cloning in animal derive from?

somatic nucleus injected to egg

Where does cloning in plantsderive from?

propagation of cutting like in roses

John B. Gurdon and his cloned frog

nucleus less egg cell from frog from UV irradiation

injected intestinal cells from tadpole

egg developed into cloned frog

Dolly sheep case

created with nuclear transfer technique using nuclease from female udder cell donor to implant to enucleated ovum.

Step by step of dolly case

udder cell (w/ nucleus) from finn dorset sheep

egg from scottish blackface with its nucleus removed

insert udder cell nucleus into enucleated egg through electro-fusion

implant embryo into surrogate blackface sheep

What is plasmid?

small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independtly

What happens when a bacterium divides? Binary fission

creates 2 new daughter cells, each carrying genetic information present in chromosome of mother cell

divides every 20 minutes

Process of recombinant DNA simplified

cut with restriction enzyme

dna fragment connected to vector with ligase enzyme

recombinant DNA trasnferred to host like E. coli

Restriction enzymes

make staggered cuts because sequences they recognize are usually symmetrical (palindromes)

What is a palindrome

base sequence that when read on 5‘ to 3’ strand is the same as when read from 3’ to 5’ complementary strand

How many nucleotides is recognition site

4-8 nucleotides

What is restriction digest?

when u cut large fragments of DNA into smaller pieces with enzyme

Ends produced when cut

Blunt - in RsaI

Sticky 5’ ends: 5’ part overhands ====-----

sticky 3’ end: 3’ part overhangs

DNA ligase

requires ATP forms Lys-NH-AMP intermediate

AMP transferred to 5’ phosphate of DNA end - activates for ligation

used to join vector with gene/DNA to be cloned forming recombinant

Transformation using heat shock method

bacterial cells treated with CaCl2 to make membrane more permeable to DNA
plasmid mixed with bacteria

cells heated at 42C for 45 seconds helping plasmid DNA enter bacterial cell

PEG/polyethylene glycol used to stabilize cell membrane and enhance DNA uptake

cells cooled to recover in nutrient media

Possible outcomes of heat shock method

bacteria take up vector + DNA fragment = recombinant plasmid/success vec+fragment

bacteria take up empty vector (no gene) = unsuccess vec
no plasmid uptake = unsuccess fragment

What does it mean when it is successful? (in plates)

example is plasmid w amp gene

plate with amp gene = recombinant DNA survives and form colonies

plate w another gene = recombinant DNA dies

What makes a good plasmid [7]

high copy number - many copies of plasmid made inside each cell

Restriction site - short region w/ restriction enzyme cuts for gene to be inserted

origin of replication/Ori - site where DNA replication starts

Antibiotic resistance gene - lets bacteria survive antibiotics

Selectable marker - identify successful transformants

promoter - sequence that starts transcription

primer binding sites - short known sequences for PCR or primers to bind

Features of naturally occuring plasmid vectors

circular, d.s.DNA molecules

size between 1000bp to 100kb

carry gene inserts up to 15kb

duplicates with host cells

What is vectors?

carriers of foreign DNA usually plasmid

has Ori, unique restriciton enzyme sites

Features in pUC19, engineered plamid vectors

ampR gene

unique citting sites

200-500 plasmids per cell to prevent overproduction and maintain control

Steps involved in molecular cloning

digest purified foreign DNA and carrier DNA (vector) cut with same restriction enzyme

digested DNA fragments fall within same size range are inserted into vectors

ligated with DNA ligase

transfer the vectors with inserts to living cells (transformation) with clones being made

cells grow on selective edia

How to know if the plasmid has an insert? (amp+ X-gal plate)

vector religate = no insert lacZ gene with multiple cloning sites so lacZ works → X-gal breaks down -→ blue

vector + insert ligation = insert in lacZ gene —> no enzyme -→ X-gal stays colourless

insert self ligates = np ampR gene/no plasmid = dies on plate so no colonies at all

beta galactosidase

an enzyme made by lacZ gene

breaks down lactose into D-glucose and D-galactose

What do we use instead of lactose in labs/

X-gal which is broken is beta galactosidase is made forming blue colonies, if enzyme disrupted by insert than colonies is white

What is alpha fragment?

portion of lacZ gene encoding first 146 amino acids on the plasmid

What is alpha complementation?

process where 2 separate fragments of the lacZ gene come together to form functional beta-galactosidase enzyme

What is replica plating?

method for transferring colonies to different plates to check their characteristics

How replica plating works?

press sterile velvet block onto master plate to pick up copies of all the colonies

press velvet block onto fresh plates one w amp, one w/o

What happens on plates for replica plating

plate w/ amp

bacteria has ampR, colony growth WILL happen

bacteria without ampR, will not have any growth (amp sensitive)

plate w/o amp

bacteria with/without ampR will have colony groups as the antibiotic is not present

Applications of recombinant DNA

therapeutic products: vaccine, antibodies, growth hormones

Diagnosis: gene therapy, CRISPR, monitoring devices

GM products: fruits, veggies, crops, microbes, animals

Energy applications: biohydrogen, bioethanol/methanol/butanol

vector constructs in gm products

two gene cassettes w/ sense strand where that gene is expressed and antisense strand where that gene is regulated or turned off

Common routes to produce transgenic plants (examples)

biological: using bacteria like agrobacterium

chemical methods: using CaCl2 or PEG

physical: microinjection, biolistic delivery/gene gune, electroporation

Protoplast…

plant cells with their cell walls removed so it is easier to work with

What is electroporation?

method used to get DNA into cells using strong electric field that creates pores in the membrane to lwt foreign DNA enter

Microprojectile bombardment/gene gun

introduce DNA into plant cells by shooting tiny particles at the cell

small gold/platinum particles coated with foreign DNA and fired at the plant cells with gene gun and penetrate cell wall

Mechanism of agrobacterium mediated transformation

Chv A and Chv B help bacterium BIND to plant cell

Acetosyringone is released to attract agrobacterium when plant is wounded or damaged

virA gene: sense acetosyringone

virG gene: once virA senses it, virG activates and turns on other genes

Vir D1 D2 C1 gene complex to nick.cut T-DNA border sequence

Vir E1 E2 binds to T-DNA forming T-complex

VirE2 D2: integrate T-DNA into plant genome

What is the Ti plasmid?

tumor inducing plasmid found in agrobacterium tumefaciens containing T-DNA region (left border n right border) and virulence regions which helps with T-DNA transfer

How Ti Plasmid works?

wonded plant releases acetosyringone

acetosyringone activates vir genes

vir proteins synthesize single stranded T-DNA

T-DNA transferred into plant

T-DNA enters nucleus and integrates

cytokinin + auxin = uncontrolled growth → tumor

or opines = special amino acids which agrobacterium can ‘eat’

GMO steps

extract embryo

gene transfer eiher by particle bombardment or agrobacterium

grow treated cell on antibiotics media

surviving callus placed in special growth media to regeneration into whole corn

keep doing and scientists select best ‘corn’/event

Flavour Savr Tomato and how it works

first GM tomato

insert copy of PG gene in antisense into tomato

this antisense mRNA bind to normal PG mRNA forming dsRNA
cells see this as abnormal and destroys it so less PG enzyme which slows down riping

in normal tomato PG is in sense strand

Roundup resistant plant

glyphosate sprayed onto GM plants

bacterial (glyphosate resistant) EPSP synthase connects shikimic acid and phosphoenol pyruvate to form EPSP

BUT if dont to normal plant, the normal EPSP synthase is blcoked which prevents plant from making amino acids

In corn

toxin producing bacterium + caterpillar = dead caterpillar

gm corn expressing toxin + caterpillar = insect resistant corn
normal corn + caterpillar = damaged corn

GM salmon

use muscle controlling gene

gene for protein coding in chinook salmon

gene for antifreeze protein in ocean pout

put into fertilised egg of atlantic salmon

Golden rice

for vitamin A deficiency

beta-carotene is vitamin A precursor = body converts beta carotene to vitamin A

many genes engineered at once

How to create a transgenic animal

gene of choice is manipulated and prepared in lab

transgene is injected into the egg of an animal

egg is implanted into surrogate

Breeding in classical technique

based on crossing and selection

similar to natural selection

having genetic barrierG

Breeding in GMO

based on random insertion

unpredictable

non natural selection

involved with 1 or 2 barriers

no barrier

Why we need to worry about GMOS

never before used

lack of knowledge on gene

not predictable

complicated systems

no long term studies

irreversible effects

Principles to evaluate GMOs

substantial equivalence: if its looks and acts the same, its safe

precautionary approach: avoid any unknown risksE

Ethical and religious considerations

Faith

Genetic info

individual right

Commercial benefit of GMOs

non tarrif barrier

influential

cost benefit

Food safety concerns

direct consumption

allergenic effects

effect on antibiotic gene

long term intake

complexity of food chain

Environmental safety concerns

change in living population

effect on food web

flow of pollen

heteroencapsidation

super weed

horizontal gene transfer

man made evolution

Flow of pollen

from insect or wind and can go up to km away