A. STEPS IN GENE TRANSFER

noisymaniacalBiotechnology

Feb 20, 2013 (4 years and 7 months ago)

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-

TYPES OF MICROBES:



a. Obligate aerobic/anaerobic



d. Facultative aerobic/anaerobic



F. BIOTECHNOLOGY: ANCIENT AND MODERN

-

SINGLE CELL PROTEIN:

a. The second largest advancement in


industrial microbiology after


penicillin production

b. In the late 1960's: Microbial cells could


be a source of proteins (dried cells of


algae, actinomycetes , fungi , yeast)

F. BIOTECHNOLOGY: ANCIENT AND MODERN


c. Microbes contain protein , CHO ,


lipids , vitamins:


1. Protein supplement in human food



2. Food Additive for flavor




3. Protein supplement for livestock

F. BIOTECHNOLOGY: ANCIENT AND MODERN

d. Algae (Spirulina) eaten by Lake Chad (Africa)


e. Requires growth of microbes on a large scale


f. SCP was not a success (need in developing




countries where no government support)

F. BIOTECHNOLOGY: ANCIENT AND MODERN


-

ANIMAL CELL CULTURES:



a.


Don't grow in suspension


b.


Interferon, enzymes, antibodies, etc..


c.


Aseptic techniques (Mycoplasmas)


d. Normal cells/ Cell lines/ Cancer cells


e.


Important cell lines (Bowes melanoma,


hybridomas, Namalwa)


F. BIOTECHNOLOGY: ANCIENT AND MODERN

Laminar flow hood

-

PLANT CELL CULTURES


a. Aseptic Techniques


b. More difficult to culture


c. Don't grow in suspension



d. Generate virus free crops


e. Regenerate whole plants from cells

F. BIOTECHNOLOGY: ANCIENT AND MODERN


-

BIOTECHNOLOGY AND GENETIC


ENGINEERING:




-

1972
-
1977: Cloning Technology





DNA Hybridization/ DNA Sequencing



A. STEPS IN GENE TRANSFER :


1.
Prepare the gene





a.

Purify chromosomal DNA from


proteins, RNA, etc…




-

BIOTECHNOLOGY AND GENETIC


ENGINEERING:



b. Cut the DNA into fragments by means of


RESTRICTION ENZYMES:

1. Their Discovery (Next slide)


2. Types (I, II, III), Properties


3. Naming (1
st

letter genus, 2
nd

two letters


species, strain, order of discovery)



A. STEPS IN GENE TRANSFER :

Restriction Enzymes


Type I and III
:


Cut
1000
nucleotides from restriction site


Methylase and Nuclease activities in one enzyme


Need ATP for enzyme activity



Type II
:


Cut at the recognition sequence


Methylase and nuclease activities are separate


Need Mg as co
-
factor




Q:
Why don’t restriction
enzymes digest chromosomal
DNA in bacterial cells?

A: Bacteria are protected from enzymatic digestion of
their DNA because some of the nucleotides in their
DNA contain methyl groups that block restriction
enzymes from digestion. Many phage do not
normally methylate their DNA, so they are
susceptible to DNA degradation by restriction
enzymes
; however, certain bacteriophages have
evolved to use methylation as a way to avoid digestion
by restriction enzymes. By attaching methyl groups to
their DNA, phage use methylation to protect their DNA
from being destroyed by restriction enzymes when they
infect bacteria.


4. Type II: 2,4, 6, 8 nucleotides



Palindromic: symmetry






Types of ends: sticky, blunt,




Incompatible cohesive



b. Cut the DNA into fragments by means of


RESTRICTION ENZYMES:

-


Sticky end enzymes: Eco RI, Bam H1 are examples





Advantage : Use same enzyme to cut vector


and DNA piece, easily retrieve


DNA





Disadvantage : Vector could anneal w/o DNA


-

BIOTECHNOLOGY AND GENETIC


ENGINEERING:


-

Blunt end enzymes : Hae III is an example








Advantage : used in making of Polylinkers





Disadvantage : more difficulty in annealing


-

BIOTECHNOLOGY AND GENETIC


ENGINEERING:

mcs




-

Incompatible cohesive termini: HindIII & XbaI





Disadvantage : have to fill in the missing


nucleotides



-

BIOTECHNOLOGY AND GENETIC


ENGINEERING:


2.


Cut the vector & Insert gene into vector


A.


Plasmids (5
-
12kb)


B. Filamentous phages


C.


Lambda Phages (10
-
20kb)


D. Cosmids (35
-
50kb)



E. Bacterial Artificial Chromosomes



F. Yeast Artificial Chromosomes








A. STEPS IN GENE TRANSFER :

What determines the choice of vector?


Insert size


Vector size


Restriction sites


Copy number


Cloning efficiency


Ability to screen for inserts



What experiments you plan?


Why need Vectors?


A.


PLASMIDS (Why are they present?):


1.

Self replicating, extrachromosomal


2.


Size: 5
-
10 kb


3.


high copy no. v.s. low copy no.


(no control, relaxed) (stringent cont.)


100
-
500 copies/cell < 10 copies/cell

Q: Why do bacteria have plasmids?

A: Bacteria possessed plasmids long before molecular biologists
dreamed of DNA cloning. Different types of plasmids exist
naturally. A primary function of plasmids is that they can
provide
bacteria with resistance to antibiotics
.



Other types of plasmids (Col
-
plasmids) can produce molecules
that
destroy other bacteria
.


Plasmids called
F
-
plasmids contain genes that produce
proteins

that can form a tube that allows for the
transfer of
plasmid DNA between bacterial cells in a process called
conjugation
.


4.

Disadvantages: limited size of insert




Only bacteria/yeast


5.

Properties of plasmids as vectors





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Steps in Gene Transfer


6. Names of plasmids





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Steps in Gene Transfer






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Tet. genes removed






copy no genes removed






Add polylinker (mcs)






Add lacZ (histochem.)



6. Names of plasmids




Lac Z codes for beta galactosidase




X
-
Gal & IPTG in media



White colonies (insert), Blue (no insert)


Steps in Gene Transfer


PHAGES: Can transfect animal cells





Larger DNA insert than plasmids


Filamentous phages: single stranded DNA phages
that infect E.coli and can be recovered as single
stranded (phage) and double stranded (plasmid)
forms. E.g. phage M13



Advantages: Useful for sequencing and mutagenesis


Steps in Gene Transfer




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lytic (cloning): temp.





packaging restrictions


cDNA and genomic libraries






Steps in Gene Transfer

PHAGES:

< 80 %

>10 %

Steps in Gene Transfer

COSMIDS: Cos sites from phage lambda






Replicate as plasmids






Up to 50 kb insert






Same packaging restrictions



3. Chose the appropriate host cell (Product yield,


activity, etc…)





-

E. coli (Prokaryote)



-

yeast (Eukaryote)




-

animal cells





-

plant cells


Steps in Gene Transfer


4. Host transformation (E. coli or yeast)



or DNA uptake by animal/plant cell


(Transfection)


Steps in Gene Transfer


Transformation of E.coli


a)


Easy to culture, replicates quickly


b)


Replicate once/ 22 min, 11 hrs get




1billion cells


c)


LB broth (yeast extract, vitamins…)


d)


Oxygen: continuous shaking



Steps in Gene Transfer



e)


Phases of growth: Lag, Log, Stat., Death


f )


10
9

transformants per microgram DNA


g)


Asceptic techniques


h)


Competent E.coli, Log phase cells


i ) Use of CaCl
2
/ ice

Transformation of E.coli



Steps: 1. streak plate/ get colonies





2. Pick colony/ grow in broth




(10
8
cells/ml


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3. Incubate in ice cells in 0.1M CaCl
2






4. Centrifuge and obtain cells (pellet)





5. Heat shock (42 C) for 90 seconds


Transformation of E.coli




6. Cool sample/ ant. resist. recovery






7. Streak again & select colonies


Transformation of E.coli


Steps: