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Chapter 9 Biotechnology and Recombinant DNA
Introduction to biotechnology
• Biotechnology – _____________________________________________________
• Used in commercial production of _______________________________________
• Pre-1980, products made by naturally occurring cells
o Naturally occurring cell found, large-scale production developed
• Post-1980, microbes, animals, plants engineered to produce array of chemicals
• Recombinant DNA, rDNA, technology – ___________________________________
o Aka ___________________________________
o Expands practical applications of biotechnology
Recombinant DNA technology
• rDNA technology employs artificial techniques to ______________________
o _________________ from any organism can be inserted into _________________
• An overview of recombinant DNA procedures
o Gene of interest inserted into ____________, usually a ________ - recombinant DNA
ƒ ___________ are used to move DNA _______________________
ƒ Must be __________________________, ie plasmid, viral genome
ƒ Vectors aka ___________________________
o rDNA is taken up by bacterium
ƒ ______________________________
o Recombinant bacterium grown in culture to form _______________
ƒ _______________________________ cells, each caries copy of rDNA
• Sometimes recombinant bacterium makes ____________________________
o Protein “harvested” by _________________________________
o _________________
ƒ Microbes grow on simple carbs
ƒ Can grow many microbes in relatively small space
o _________________
ƒ Doesn’t require toxic chemicals, high temps and pressure
ƒ Doesn’t produce toxins hazardous to workers
o _________________
ƒ Bacteria grow to high yields, very fast
• Sometimes “gene of interest” is product
o Recombinant bacterium used to _____________________________________
o Gene can be used for:
ƒ Engineered into plants, other bacterium
ƒ Various different purposes
Tools of biotechnology
• Restriction enzymes
o Special class of ____________________________
o Occur naturally in bacteria
o Enzymes recognize and cuts DNA at ___________________ sequences
ƒ Cuts DNA _______________________ each time
•
•
o Many make “staggered cuts” or produce “sticky ends”
ƒ Can bind to complementary pieces of single stranded DNA
ƒ Identical sticky ends can be combined easily - ________________
ƒ Covalently linked by enzyme _______________________
Vectors
o DNA vectors need to have certain properties
ƒ
• All recombinant vectors need to be replicated at some point
ƒ
• _________________ vectors more easily manipulated
• _________________ vectors more fragile
ƒ
• Circular form is important in __________________ of plasmid
• Viral DNA inserts into genome quickly
ƒ
• Gene that allows for selection of recombinant organisms
• Often antibiotic resistance gene
o Shuttle vectors – plasmids capable of existing in ____________________________
ƒ Can even exist in both eukaryotes (yeast) and bacteria
o __________________________ can generally transfer larger pieces of DNA
o Choice of vector depends on ______________________ organism
Polymerase Chain Reaction
o _______________________ samples of DNA quickly in a test tube
ƒ From one piece of DNA, can make billions of copies in few hours
o Can amplify ___________________ pieces of DNA
o Can only be used to amplify relatively ___________________ pieces of DNA
o PCR can be applied to any situation that requires amplification of DNA
ƒ Detect presence of pathogens from small sample
Techniques of genetic modification
Inserting foreign DNA into cells
• rDNA techniques require that DNA molecules be inserted into cells
•
o Take up of DNA from surrounding environment
o Many bacteria do not naturally transform
o Simple ____________, ______________ treatments can make bacteria competent
•
o Uses __________________________ to form microscopic _________ in membrane
ƒ DNA enters cells through pores
•
o Shoots DNA coated onto ________________________ into cell
ƒ Bursts through cell wall
o Some of the DNA inserts into genome
•
o Cell walls removed __________________________ - protoplast
o Protoplasts in solution able to fuse at low frequency
o DNA from hybrid cells undergo ___________________________
o Important in manipulating ________________________________
•
o Use of tiny syringe to __________________________ inject DNA into cell
ƒ Needle punctures the plasma membrane
• After DNA is introduced, still needs to either exist on self-replicating vector or insert into
genome
Making a gene product
• Gene products are frequently object of genetic engineering
• Various organisms employed
• E. coli
o Easily ___________, familiar with _______________
o Genes can be controlled using many different inducible promoters
o However, E. coli produces ______________________ as part of cell wall
ƒ Can contaminate final products
o Doesn’t _________________ gene product
ƒ Cells need to be ____________________ and purified from solution
ƒ Can be expensive on industrial scale
• S. cerevisiae
o Best understood ______________________________________
o Yeasts carry many easily manipulated, transferable plasmids
o More successful in expressing ________________________ genes than bacteria
o Yeasts can _____________________ many of their products
• Mammalian cells
o Often best suited for making protein products for ________________________
ƒ Cells secrete products
ƒ Low risk of _____________, __________________
o Manipulation of DNA is ______________________ as bacteria, yeasts
• Plant cells
o Useful for producing ________________________
ƒ Codeine (painkiller)
ƒ Melanin (pigment used in sunscreens)
o Advantages over mammalian cells include
ƒ Relative ___________________ of large-scale productions
ƒ Low risk of contamination by mammalian pathogens, ______________ genes
o Manipulation of DNA is ______________________ as bacteria, yeasts
Applications of rDNA
Therapeutic applications
• Obtaining insulin from animals is ______________, not as _____________ as human insulin
o Production of ______________ was first major breakthrough of rDNA technology
o Many other hormones being produced
• Subunit vaccines – vaccine consisting of ______________________ of pathogen
o Made by genetically modifying __________________
ƒ Eg hepatitis B
o Advantage of subunit vaccine, no chance of _____________________ vaccine
• DNA vaccines – plasmids that include __________________
o Plasmids cloned in ____________________
o HIV, SARS, influenza being tested
• Artificial blood – prepared using human __________________________
o Can be used for __________________________
• Gene therapy – curing genetic disease by replacing ___________ gene with _________ gene
o _____________ used as vector to introduce normal gene
o Used successfully in past to treat hemophilia, SCID
o Still very preliminary
• Gene silencing – “switching off” of a gene
o Natural defense against viruses, transposons
o Use ______________________, _________
ƒ Double stranded short interfering RNAs, siRNA target particular gene
• ie virus gene
ƒ siRNA bind to mRNA, causing enzymatic destruction
• Expression of gene has been silenced
o RNAi has inhibited hepatitis B virus
The Human Genome Project
• Major international project that sequenced the entire human genome
o Took about 13 years
o About ____________________ base pairs
o ________________ to _____________ genes
• 98% of DNA is _____________________ – “_____________ DNA”
• Current step is to locate specific genes, determine function
Scientific applications
• rDNA technology has more uses than making products
• rDNA can be used to make many copies of DNA, applied to many other uses
• DNA sequencing - determination of exact ________________________ in DNA
o Most common technique to day is _______________________________
ƒ ____________________ are sequenced, and pieced together by computer
ƒ Gaps are then filled in
ƒ Relatively _________________, but ________________
o Small genomes easy to sequence nowadays
• Bioinformatics – science of understanding _____________________________________
o Abundance of gene information, bioinformatics attempts to organize it, rationalize it
• Proteomics – science of studying all the ___________________________
o Important to know what, when, how genes are expressed
• Southern blotting – technique to identify ______________________
o Can look for presence of mutated genes
o Process:
ƒ DNA is digested using ______________________________
ƒ Digested fragments are separated by ___________________
• Gel electrophoresis
• Fragments called _______, restriction fragment length polymorphisms
ƒ _______________ transferred onto filter by blotting
ƒ Filter exposed to radioactive probe
• Probe specific for gene
ƒ Fragments identified by exposing filter to film
• “Takes a picture”
• DNA sample can be tested for presence of gene
o Genetic screening uses Southern blots to screen for ________________________
• Forensic microbiology
o DNA probes used to rapidly identify __________________
ƒ Also be applied to find microbes in soil, environmental samples
o DNA fingerprinting – use of _____________ to identify specific strains of microbes
ƒ _______________________ identification
ƒ Used to track bacterial isolates during an outbreak of a disease
Agricultural applications
• Selecting genetically desirable plants through breeding time consuming, laborious
• Today, desired plants genetically engineered
• Recombinant DNA introduced into plants using several methods
o Protoplast fusion
o Gene gun
o
ƒ Very clean, elegant
ƒ Uses plasmid, ________________, from Agrobacterium tumefaciens
ƒ Infects plants, causes ________________
ƒ Part of infection is to insert bacterial DNA into _________________
• Bacterial genes stimulates ____________________
ƒ Scientist have engineered Ti plasmid so that it _________________________
• Carries ____________ genes instead of _______________ genes
• Eg, pesticide resistance, pest resistance (Bt corn)
Safety issues and the ethics of using rDNA
• Always concerns about safety of new technologies
o Impossible to prove any technology is 100% safe
• Genetic engineering can produce higher crop yields, use less pesticides
• Genetic screening can help find diseases to start early treatment
• Gene therapy can cure incurable genetic diseases
• But ….
• What is toxicity of rDNA?
o Are protein products safe?
• What if pesticide resistance spread to weeds?
o How to control their growth now?
o Similar to spread of antibiotic resistant bacteria
• Does it harm environment?
o Do pest resistant crops also kill nonpests?
• Who has control of information of genetic screens?
o Do you want life insurance companies, you work to know?
• How safe is gene therapy?
o Will it cause more problems than cures?