Download Chapter 9, part A

TORTORA • FUNKE
• CASE
Microbiology
AN INTRODUCTION
EIGHTH EDITION
B.E Pruitt & Jane J. Stein
Chapter 9, part A
Biotechnology and Recombinant
DNA
Biotechnology and
Recombinant DNA
• Biotechnology:
– The use of microorganisms, cells, or cell
components to make a product
– Foods, antibiotics, vitamins, enzymes
– Old science - cheese, beer, wine and bread
• Recombinant DNA Technology:
– Insertion or modification of genes to produce
desired proteins
Figure 9.1.1
Figure 9.1.2
Table 9.1.1
Table 9.1.2
Tools of Biotechnology
• Enzymes that do the work:
–
–
–
–
Restriction enzymes
DNA polymerase
DNA ligase
Reverse Transscriptase
• Vectors to carry DNA
– Plasmids
– Viruses
• DNA sources
– Synthetic
– Gene libraries
• Important procedures
– PCR
– Blotting Southern / northern / western
Tools: Restriction Enzymes
• Cut specific sequences of DNA
– Palindromes: Madam I’m Adam; Race car; wow
and mom
• In wild cells they at as a type of disease
defense by destroying bacteriophage DNA in
bacterial cells
• Cannot digest (host) DNA with methylated
cytosines
• May cut DNA blunt or with “sticky ends”
Figure 9.2
Vectors
• Carry new DNA to desired cell
• Shuttle vectors can exist in several
different species
• Plasmids and viruses can be used as
vectors
Vectors
Figure 9.3
(PCR)
• To make multiple copies of a piece of DNA
enzymatically
• Used to
– Clone DNA for recombination
– Amplify DNA to detectable levels
– Sequence DNA
– Diagnose genetic disease
– Detect pathogens
PCR
Figure 9.4.1
PCR
Figure 9.4.2
DNA can be inserted into a cell by:
• Transformation
• Electroporation
• Protoplast
fusion
• Microinjection
• transduction
Figure 9.5b
DNA can be inserted into a cell by:
• Microinjection
• Gene gun
Figure 9.6 & 7
Obtaining DNA
• Gene libraries are made of pieces of an
entire genome stored in plasmids or
phages
• cDNA is made from mRNA by reverse
transcriptase
– Why???
• Synthetic DNA is made by a DNA
synthesis machine
Selection & Mutation
• Selection: Culture a naturally-occurring
microbe that produces desired product
• Mutation: Mutagens cause mutations that
might result in a microbe with a desirable
trait
• Site-directed mutagenesis: Change a
specific DNA code to change a protein
• Select and culture microbe with the desired
mutation