Lecture 12 - Genetic Manipulation I

Question 1

genetic manipulation is interpreted as:

Answer 1

the cloning, modification and analysis of DNA sequences

Question 2

what does genetic manipulation allow us to do?

Answer 2

• produce proteins (i.e: for detailed analysis in structural biology)
• explore the function of genes and pathways (human health)
• identify species and biodiversity

Question 3

cloning a specific gene allow us to:

Answer 3

• allows specific sequencing of the gene
• allows expression of the protein product in, for example, bacteria
• allows for the modification of a gene to produce an altered protein

Question 4

the first application of recombinant DNA technology:

Answer 4

bacterial production of human insulin in bacteria (1978 - licensed in 1982)

Question 5

modifying a whole organisms DNA to explore the consequences allows us to:

Answer 5

• allows analysis of gene function through phenotype of a knockout
• allows the determination of how gene expression is controlled
• allows modification of the gene to produce an altered protein and explore the phenotypic consequences

Question 6

first inherited modification of a mammalian genome:

Answer 6

1981

Question 7

examples of genetic manipulation in ecology and biodiversity:

Answer 7

• allows analysis of population movement
• allows identification of species in an environment
• allows the characterisation of microbes that cannot be cultured

Question 8

an outline of the gene cloning procedure:

Answer 8

(1) the insertion of a fragment of DNA (made through e.g: restriction endonuclease) into a cloning vector (joining: ligation)

(2) the subsequent propagation and amplification of the recombinant DNA molecule in a host organism (each cell grown as a clone)

Question 9

Cloning allows the ____________ a gene (or other DNA sequence) in a way that allows it to be __________ – for _________ and ____________

Answer 9

(1) purification

(2) replicated

(3) analysis

(4) modification

Question 10

difference between the purification of an enzyme and a gene:

Answer 10

• enzymes can be purified from cells and their properties can be studied using biochemistry and structural biology, however they can not be replicated and amplified like DNA fragments

Question 11

why is the cloning of a pure gene sample separated from the mass of an organisms DNA useful?

Answer 11

• allows the DNA sequencing and analysis of features
• allows genetic manipulation in order top change amino acid sequence in a genes subsequent protein
• allows fusion of a gene to a reporter protein such as GFP
• allows gene over-expression for desired protein production
• can help assign structural features of a protein to function

Question 12

the basics you need to go about cloning a gene:

Answer 12

(1) source of DNA that is to be cloned

(2) a cloning vector to act as the carrier of the DNA being cloned

(3) a way of cutting the source DNA into appropriate size (usually a restriction enzyme) and linearizing the vector

(4) a method for joining DNA fragments together

(5) a method for joining DNA into E.coli cells

Question 13

cloning vector:

Answer 13

a DNA molecule, capable of replication in a host organism, into which a DNA sequence [gene] is inserted to construct a recombinant DNA molecule

Question 14

common features of cloning vectors:

Answer 14

• replicate independently of and at a higher level than the host chromosome: aiding purification
• contain a selectable marker (e.g: antibiotic resistance gene) - allowing fore identification and selection of vector containing host bacteria
• contain specific restriction enzyme recognition sites (cloning sites) allowing linearisation of circular DNA sequences into the vector
• usually derived from naturally-occuring plasmids and bacteriophages

Question 15

most common vectors:

Answer 15

bacterial plasmids

Question 16

characteristics of natural plasmids:

Answer 16

• extrachromosomal
• mostly circular (some linear)
• 1kb-100kb in size
• non-essential
• usually carry ‘supplemental’ genetic information, but may be cryptic
• often confer selective advantage on the host bacterium

Question 17

antibiotic resistance plasmids code for:

Answer 17

• encode for proteins that provide resistance to antibiotics

Question 18

sex plasmids allow for:

Answer 18

sex plasmids allow for the exchange of DNA between bacteria

Question 19

function of col plasmids:

Answer 19

col plasmids encode for various toxins that kill neighbouring cells

Question 20

degradative plasmids:

Answer 20

degradative plasmids encode for a variety of enzymes that allow the metabolism of unusual substances

Question 21

virulence plasmids:

Answer 21

virulence plasmids generally encode proteins that turn a bacterium into a pathogen

Question 22

characteristics of bacteriophages as vectors:

Answer 22

• can be either circular of linear DNA
• 5kb to hundreds of kb in length
• infect the host, replicate and package DNA, then kill the cell
• some viruses can integrate into the host chromosome and lay dormant

Question 23

plasmid cloning vectors have been engineered so that they are small and contain (+ what do they also sometimes contain?):

Answer 23

• a replication origin
• a selectable marker
• suitable cloning site(s)

sometimes they also contain:
- a multiple cloning site (MCS)
- a method for detecting recombinant molecules

Question 24

the selectable marker:

Answer 24

• usually an antibiotic resistance gene such as the b-lactamase gene (ampicillin resistance)

Question 25

examples of plasmids (1) - pBR327:

Answer 25

• replication of origin (Ori): derived from natural plasmids of the “ColE1” family, promotes replication initiation by host replication enzymes
• restriction enzyme sites: engineered to contain several unique sites including Pstl, EcoRI, HindIII, Al/I - which can all be used for cloning
• has two selectable markers derived from native plasmids: (1) ApR (ampicillin resistance) (2) TcR (tetracycline resistance)

Question 26

examples of plasmids (2) - pBluescript SK+:

Answer 26

• ampicillin resistance gene
• origin of replication is deregulated for high copy number (500/cell)
• lac promoter and lacZa gene
• MCS multiple coding sites
• bacteriophages origin to make ssDNA (doesn’t have other proteins for phage production

Question 27

restriction enzymes:

Answer 27

• usually cut a palindromic (e.g symmetrical) sequence
• derived from a wide variety of (usually) bacterial sources
• can generate ‘sticky’ ends which are resealable with DNA ligaments: [3’ OH] & [5’ Phosphate]

Question 28

in what two ways can restriction enzymes cut?

Answer 28

restriction enzymes can cut both sticky and blunt ends

Question 29

different restriciton enzymes sometimes produce the same sticky ends:

Answer 29

this means that certain fragments can be legates into vectors that are digested with BamHI - something that is particularly useful when making a genomic library

in some instances this means that the resulting hybrid site can generally not be cut again

Question 30

origin of restriction enzymes:

Answer 30

• mainly derived from bacteria that are protecting themselves from viral invasion
• a some of the “rare cutters” (i.e: >8 base cutters) are derived from single cell eukaryotes and are involved in various aspects of biology such as mating types in yeast

Question 31

why is bacterial DNA not degraded?

Answer 31

this is because bacterial DNA is often methylated

Question 32

insertional inactivation: lacZa and Blue-white screening:

Answer 32

•The b-galactosidase is a large enzyme encoded by lacZ (~1000 amino acids)

•LacZ enzyme can be split into 2 peptides – a small fragment called LacZa and large fragment called LacZW

•Neither LacZa or LacZW are active alone – BUT they can combine to form active enzyme

•Introduction of a plasmid expressing LacZa into a strain that already expresses LacZW will lead to functional b-galactosidase activity

•Active b-galactosidase can convert the colourless substrate X-gal into a BLUE product

Question 33

natural “transformation”:

Answer 33

• bacteria can take up DNA from the environment, usually from other dying organisms
• some bacteria are naturally competent (i.e: can constitutively take up DNA)
• some bacteria are inducibly competent. This means they can become competent under particular growth conditions such as stress

Question 34

hypotheses to why cells competent:

Answer 34

• H1: a primitive form of “sex” to aid gene diversity
• H2: DNA as a food, simply using resources available
• H3: helps to repair DNA, particularly under stressed conditions
• H4: allows horizontal gene transfer which gives an evolutionary advantage

Question 35

making E.coli cells competent artificially:

Answer 35

• E.coli cells are grown to mid-log phase
• they are harvested by centrifugation
• then they are incubated in ice cold CaCl2

this results in the cells to take up DNA