gene cloning and fusion protein expression

Question 1

what does DNA cloning do

Answer 1

DNA cloning yields multiple copies of a DNA segment or gene

Question 2

general features of gene cloning

Answer 2

restriction enzymes, plasmids and use of bacteria

Question 3

Type II restriction enzymes

Answer 3

recognise specific nucleotide sequences in DNA
cut both strands of the DNA molecule

Question 4

how long can the recognition sequence be for RE

Answer 4

4,5,6,7 or 8 nucleotides long

Question 5

do RE recognise all sequences

Answer 5

no different RE recognise different sequences in DNA

Question 6

what do RE produce

Answer 6

staggered cuts in DNA
leave short ss “tails” at the ends of DNA fragments
produce blunt-end DNA fragments no over hangs or unpaired bases

Question 7

what are ss tails known as ?

Answer 7

cohesive or sticky ends

Question 8

plasmids

Answer 8

are molecules of DNA that are found in bacteria separate from the bacterial chromosome

Question 9

what are the essential features of a plasmid for cloning ?

Answer 9

1. ORI
2. unique restriction site- only occurs once
3. small size
4. selectable marker

Question 10

what does the selectable marker do?

Answer 10

provides resistance to antibiotic

Question 11

what enzyme does ampicillin resistance gene code for

Answer 11

beta-lactamase

Question 12

what does beta lactamase catalyse

Answer 12

the hydrolysis of ampicillin

Question 13

what is the result of the ampicillin resistant gene

Answer 13

destroys the antibiotic
allows bacteria to grow

Question 14

what is a plasmid called in gene cloning ?

Answer 14

cloning vector

Question 15

what can a cloning vector do?

Answer 15

carry foreign DNA into a host cell and replicate there

Question 16

naturally occuring plasmids have been genetically engineered to possess what ?

Answer 16

features for use as cloning vectors

Question 17

what is a polylinker/ multiple cloning site

Answer 17

a synthetic DNA fragment with restriction sequences for several RE can be inserted into a plasmid

Question 18

what does gene cloning use bacteria for?

Answer 18

to produce many identical copies of the same recombinant molecule

Question 19

what are the steps of cloning ?

Answer 19

1.vector + DNA
2.recombinant DNA
3.transform host cells
4.select for transformed cells
5.screen host cells that contain recombinant molecule

Question 20

what do RE and DNA ligase allow

Answer 20

insertion of DNA fragments into cloning vectors

Question 21

experiment: to clone the gene X (350bp) into Eco RI site of the vector pBR322

Answer 21

1. digest the vector with EcoRI
2. digest the Gene X with EcoRI
   (cut ends are sticky and complementary)
3. mix digest vector and gene together in an eppendorf
4. add DNA ligase to mixture in eppendorf
5. leave mixture at 37 degrees for 20 mins
   result: pBR322 religated and recombinant molecule

Question 22

DNA ligase

Answer 22

catalyses the formation of the 3’-5’ phosphodiester bond

Question 23

Recombinant DNA

Answer 23

DNA from two sources is incorporated in vitro into a single molecule

Question 24

bacterial transformation

Answer 24

transfer of plasmid/recombinant molecule into bacteria
phospholipid bilayer of the PM has a hydrophilic exterior and hydrophobic interior
DNA unable to freely pass through the membrane

Question 25

artificial: Calcium chloride transformation

Answer 25

CaCl2 treatment of bacterial cells produces competent cells which will uptake DNA after a heat shock set up

Question 26

how does a colony represent a clone

Answer 26

all cells in the colony are descendants of one cell

Question 27

To identify correct clone

Answer 27

isolate the plasmid DNA from a number of transformants analyse plasmids using restriction enzyme digestion agarose gel electrophoresis

Question 28

isolate the plasmid DNA from a number of transformants

Answer 28

1 inoculate colony into broth 2 grow overnight @37 degrees 3 spin down 1.5ml 4 lyse cells 5 extract plasmid

Question 29

restriction enzyme digestion of extracted DNA with EcoRI

Answer 29

restriction digest: add 10ul extracted DNA to eppendorf add 2ul buffer add 1ul EcoRI 7ul water leave mixture at 37 degrees for 60 mins

Question 30

agarose gel electrophoresis

Answer 30

used to separate charged molecules DNA is neg charged DNA is moved by an electric current through a matrix of agarose large pieces move slower than small pieces of DNA

Question 31

gene cloning and expression is used to produce protein drugs

Answer 31

1.foreign DNA inserted into plasmid, recombinant plasmid inserted into a bacterial cell 2. reproduction in bacterial cell results in cloning of the plasmid including the foreign DNA 3. results in production of multiple copies of a single gene 4. cloned genes expressed to produce a protein

Question 32

examples of gene cloning to produce protein drugs

Answer 32

insulin- diabetes interferon- hepatitis C/ cancer factor VIII- haemophilia human growth hormone tissue plasminogen factor (TPA)- dissolves blood clots

Question 33

what are expression vectors

Answer 33

plasmids that have been modified for the purpose of expressing high levels of a foreign protein in bacteria

Question 34

what have expression vectors been engineered to contain?

Answer 34

appropriate sequences for transcription, translation and protein purification

Question 35

how do expression vectors produce a protein ?

Answer 35

production of large amounts of mRNA which can be translated into protein produce protein using the host cells protein synthetic machinery

Question 36

features of an expression vector

Answer 36

ORI unique restriction site small size selectable marker promoter RBS tag protein to aid purification

Question 37

how to get optimal induction time

Answer 37

induce large vol of culture

Question 38

affinity chromatography

Answer 38

purification of the induced protein

Question 39

how are fusion proteins purified

Answer 39

from bacterial lysates by passing the lysate through a column containing resin to which the fusion protein can bind other cellular proteins will flow through the column after washing the fusion protein can be released from the column

Question 40

what is the purification of a fusion protein analysed by

Answer 40

SDS-PAGE

Question 41

What is used to estimate the molecular weight of a purified protein

Answer 41

protein molecular weight markers

Question 42

explain the difference between "sticky ends" and "blunt ends"

Answer 42

Sticky ends: short, ss stranded overhangs left at the ends of DNA fragments after being cut by RE blunt ends: are DNA fragments with no overhangs or unpaired bases

Question 43

what is a polylinker

Answer 43

a synthetic DNA fragment with restriction sequences for several different restriction enzymes

Question 44

why is a polylinker useful in cloning ?

Answer 44

provides multiple options for inserting foreign DNA into the plasmid

Question 45

how are bacterial cells made "competent" for transformation?

Answer 45

made competent for transformation through artificial methods like Calcium chloride treatment, which helps them uptake DNA after a heat shock step.

Question 46

purpose of using a selectable marker, such as ampicillin resistance gene in cloning?

Answer 46

allows for the selection of transformed cells by providing resistance to an antibiotic, so only cells that have taken up the plasmid can survive and grow in the presence of an antibiotic

Question 47

what is a fusion protein ?

Answer 47

a protein that results from the expression of recombinant DNA, typically where a protein of interest is linked to a tag protein

Question 48

how is a tag protein used in the purification of a fusion protein

Answer 48

through affinity chromatography