MicrobiologyAN INTRODUCTION

TORTORA • FUNKE • CASE

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

• 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”

Tools: Restriction Enzymes

Figure 9.2

• Carry new DNA to desired cell

• Shuttle vectors can exist in several different species

• Plasmids and viruses can be used as vectors

Vectors

Vectors

Figure 9.3

• 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

Polymerase Chain Reaction (PCR)

PCR

Figure 9.4.1

PCR

Figure 9.4.2

• Transformation

• Electroporation

• Protoplast fusion

• Microinjection

• transduction

DNA can be inserted into a cell by:

Figure 9.5b

• Microinjection

• Gene gun

DNA can be inserted into a cell by:

Figure 9.6 & 7

• 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

Obtaining DNA

• 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

Selection & Mutation