MI2011: Biotech

Question 1

Vectors

Answer 1

tools for inserting DNA into cells for sequencing or expression

Question 2

What are two types of vectors that are manipulated

Answer 2

Bacteriophage vectors and Plasmid vectors

Question 3

Bacteriophage vectors

Answer 3

• can use in vitro packaging in which you add your DNA, phage heads and tails and enzymes
• results in infectious virus particles capable of injecting your DNA into a susceptible host cell

Question 4

Plasmid vectors

Answer 4

• use restriction endonucleases to cut DNA at specific sties, use DNA ligase to re-join
• T-vector cloning for PCR products
• Induce competency in bacterial cells, uptake the plasmid by transformation

Question 5

Shuttle vectors

Answer 5

can replicate and be stably maintained in 2 or more unrelated hosts

Question 6

Expression vectors

Answer 6

include regulatory sequences to switch on and off expression of inserted genes

Question 7

Phage vectors

Answer 7

phage genome allows for holding larger amounts of DNA than a plasmid

Question 8

Cosmid vectors

Answer 8

specialized plasmids that use phage virions

Question 9

Artificial chromosomes

Answer 9

• bacterial artificial chromosomes are engineered from conjugative plasmids
• yeast artificial chromosomes, replicate in yeast like normal chromosomes and allow a large DNA fragment insertion

Question 10

Mutagenesis

Answer 10

• mutates a gene at a specific point
• hybridises a mismatched oligonucleotide
• oligo acts as primer for synthesis
• new DNA strand contains mutant gene
• Useful for: protein engineering; constructs proteins with new properties

Question 11

Recombinant expression: Protein expression

Answer 11

Insert genes into vectors which have promotors that result in expression of the gene. Result: production of specific protein

Question 12

Transfection

Answer 12

Introduce plasmid into eukaryotic cells for expression of protein

• transient (plasmid not integrated into chromosome)
• stable (plasmid integrates)

Question 13

Describe how a colony containing a plasmid with an insert is selected

Answer 13

Blue-white screening

Question 14

List some examples of therapeutics

Answer 14

• Immunomodulators
• Hormones (insulin)
• Enzymes
• Growth Factors
• Vaccines
• Gene therapy

Question 15

What bacteria is typically used as a cloning host in eukaryotes

Answer 15

Saccharomyces cerevisiae

Question 16

What bacteria/s are used in cloning hosts for prokaryotes

Answer 16

E.coli and Bacillus Subtilis