10/3/2016 1 Biotechnology & DNA Technology Chapter 9 BIO 220 Biotechnology • Is the use of microorganisms, cells, or cell components to make a product • By inserting, deleting, or modifying genes with recombinant DNA (rDNA) technology microbes are being used as “factories” to produce chemicals they would not ordinarily How to make recombinant DNA Fig. 9.1 Vectors • i.e. plasmids or viruses • Must self-replicate (needs origin of replication) • Selectable marker (i.e. antibiotic resistance) • Restriction enzyme sites Fig. 9.3
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Biotechnology & DNA Technology
Chapter 9
BIO 220
Biotechnology
• Is the use of microorganisms, cells, or cell
components to make a product
• By inserting, deleting, or modifying genes with
recombinant DNA (rDNA) technology
microbes are being used as “factories” to
produce chemicals they would not ordinarily
How to make recombinant DNA
Fig. 9.1
Vectors
• i.e. plasmids or viruses
• Must self-replicate (needs origin of
replication)
• Selectable marker (i.e. antibiotic resistance)
• Restriction enzyme sites
Fig. 9.3
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Restriction enzymes
Fig. 9.2
How to make recombinant DNA
Fig. 9.1
Polymerase Chain Reaction (PCR)
Fig. 9.4
Denature
Anneal
Elongation
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Some applications of PCR
• Detection of pathogens (i.e. Mycobacterium
tuberculosis)
• Mycology and parasitology
• Viral characterization
• Dentistry
• Environmental microbiology
We made the recombinant plasmid, now . . .
Fig. 9.1
Inserting foreign DNA into cells
• Conjugation
– Plasmids transferred between closely related
microbes
• Transformation
– Cells must be made “competent” in order to take
up plasmids
• Electroporation
– Cell walls must usually be digested (protoplasts)
Gene gun
Fig. 9.6
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Microinjection
Fig. 9.7
Big picture
• There are a number of ways to get foreign
DNA into a variety of cell types. However, the
foreign DNA will only survive if it is (1)
incorporated into a self-replicating vector, or
(2) incorporated into one of the cell’s
chromosomes by recombination.
The rDNA is in the cell, but . . .
Fig. 9.1
How do we find the cell with the gene of
interest?
• Blue-white screening
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Colony hybridization – Is it really the gene
of interest?
Fig. 9.12
Now what?
Fig. 9.1
We have made the gene product . . . We have made the gene product . . .
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