Transcript Document

Bridge Class
Routing vectors to Genetic
Engineering
Introduction to Vectors
Defining the purview of genetic engineering?
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Identification of genomic sequences
• Reverse/Forward genetics
Isolation of genomic sequences
• PCR, RT-PCR, genomic/cDNA libraries
Verification of genomic sequences
• Sequencing technologies
Manipulation of genes and genomic sequences
• Cutting, pasting, changing (Site directed mutagenesis)
• Tools: Enzymes
Assorting combinations from unrelated species
• Tools: Enzymes
Cloning of rDNA
• Making use of vectors and strains
Expressing heterologous biomaterial (proteins of therapeutic or
economic value in microorganism to provide proof of concept,
pilot scale, industrial level
Considerations prior to cloning
(Plasmid Vectors)
PCR amplified
Forced ‘A’
tailing
Pfu
polymerase
Source of the
genomic fragment
Released thru
Restriction
(frm another
vector)
Taq
polymerase
• Linearaisation of
vector with
compatible RE
•CIAP Treatment of
vector
Choice w.r.t
Ligation strategies
TA Cloning
TOPO-TA
Cloning
S1
nuclease
Blunt end
Cloning
•Linkers and adaptors
•Homopolymeric tailing of
vector and insert
Cohesive end
Cloning
Standard methodology of cloning
Pipeline downstream of Ligation
Transformation
•Heat shock method
•Electro-transformation
Preparation of Host:
Competent cells
(Chemi compis or electro-compis)
•CaCl2 method (Mandal and Higa, 1970)
•RbCl2 Method (Hanahan, 1983)
•Other protocols
(eg For electro-competent, glycerol)
Ligation mix (mixture of
recombinant and nonrecombinant plasmids)
A competent E. coli strain
E.coli Transformation: Introduction of DNA into host cells
Classical definition: Natural uptake of naked ds DNA by bacterial cells.
•Fred Griffiths (1928) Streptococcus pneumoniae (a.k.a. Pneumonococcus or
Diplococcus)
•Avery, McCarty and MacLeod (1944) proved that DNA is the transforming principle .
Competence
•Only competent cells can take up DNA from external mileu
•Competent state induced in response to nutritional shut down
•Not all bacteria are competent, all the time (E. coli not naturally competent)
Natural
competence
Artificially
induced
E. coli K12: laboratory host for rDNA
Ensuring Safety in Molecular Biology laboratory/human health/ environment
Outcome of Asilomar Conference (Center on California’s Monterey Peninsula
1976)
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Scientific fraternity realizing the importance of working with “safe” strains
of bacteria, viruses and vectors
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Containment to be made a part of expt design, level of containment
proportional to risk involved/biohazard (Risk assessment and analysis)
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NIH formed the Recombinant DNA Advisory Committee (RAC)
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It took 1 year (1976) before the first “safe” (EK2 category) line of E. coli
was released
That year, RAC released a set of guidelines requiring the use of safe
bacteria