2. Genetic modification of microbes

Question 1

Types of genetic variation

Answer 1

Mutation
Recombination

Question 2

Random mutagenesis

Answer 2

Random mutations caused by exposure to a mutagenic agent

Question 3

Types of mutagens

Answer 3

Physical: radiations
Chemical
Biological: transposons

Question 4

Types of mutagenesis by recombination

Answer 4

Sexual reproduction (eukaryotes)
Protoplast fusion

Question 5

Protoplast fusion

Answer 5

Fusing paternal and maternal cells to create a mutant

Question 6

Genetic engineering

Answer 6

Introducing mutations using DNA technology

Question 7

Main steps of recombinant DNA technology

Answer 7

Genetic engineering
Incorporation of recombinant DNA into the cell
Selection of clones

Question 8

Methods for introduction of DNA into cells

Answer 8

Transformation
Electroporation
Conjugation
Transduction

Question 9

Origin of replication

Answer 9

Particular sequence in a genome at which replication starts

Question 10

Expression in cis

Answer 10

A cis gene regulates expression of genes on the same DNA

Question 11

Expression in trans

Answer 11

A trans gene regulates expression of genes on separate DNA

Question 12

Example of regulation in cis

Answer 12

Operons

Question 13

Example of regulation in trans

Answer 13

Plasmids

Question 14

Process for introducing a directed deletion in a genome

Answer 14

Deleting a fragment of the coding sequence in a separate plasmid
Opening the target DNA and plasmid with the same restriction enzyme
Ligating the ends of the plasmid with target DNA

Question 15

Process for introducing a point mutation in a genome

Answer 15

Synthesis of two sets of DNA primers containing the mutation in its center
Extension with DNA polymerase
Ligation of the two PCR products
Extension
Introduction by allelic exchange

Question 16

Advantages of using eukaryotes as hosts for expression of human DNA

Answer 16

Their genetic machinery is similar to ours

Question 17

How do you check if your vector was inserted correctly?

Answer 17

You include antibiotic cassettes or metabolic markers into the vector

Question 18

Metabolic markers

Answer 18

Genes that encode an enzyme that is essential for growth of the organism in the culture medium

Question 19

Process of selection with X-gal

Answer 19

Vector carries part of the gene on each end of the MCS
If the insert was introduced, that gene will be broken (because the insert will be inside it) and the enzyme will not be expressed (white colonies)
If the insert was not introduced, the enzyme is expressed (blue colonies)

Question 20

Expression vector

Answer 20

Plasmid designed for protein expression in cells. It contains a specific gene and uses the cell’s mechanisms for protein synthesis

Question 21

Features common to all vectors

Answer 21

Origin of replication
Selectable marker
Multicloning site

Question 22

Elements necessary for protein expression

Answer 22

Transcription: promoter, termination sequence
Translation: RBS, start codon, termination codon

Question 23

Inducible promoter

Answer 23

Promoter whose activity is induced by the presence or absence of a particular substance in the medium

Question 24

Utility of peptide tags

Answer 24

Ensure that protein is not degraded or misfolded
Facilitates detection and purification of product

Question 25

Linker sequences

Answer 25

Short sequences included in the peptide tag to allow us to remove the tags

Question 26

Methods for purification using tags

Answer 26

Anti-tag antibodies bind the tag
If the tag is maltose binding protein, we use maltose to bind the tag
Glutathione S transferase (GST) tagged proteins are purified with glutathione
Polyhistidine tails can be detected and retained in a column

Question 27

Periplasmic or external secretion signal

Answer 27

Signal peptide that induces or improves product secretion

Question 28

Most commonly used types of yeast

Answer 28

Saccharomyces cerevisiae
Pichia pastoris

Question 29

Types of cloning vectors derived from yeast

Answer 29

YEP
YAC