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Molecular Genetics > Lab Practical > Flashcards

Lab Practical Flashcards

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1
Q

purpose of vector

A

used to carry foreign DNA fragments into host cells

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2
Q

role of promoters

A

reg gene expression

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3
Q

markers

A

aid in selection of transformed cells

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4
Q

tags

A

facilitate protein purification/ detection

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5
Q

what is molecular cloning

A

set of experimental methods used to construct recombinant DNA molecules that can be replicated in suitable host organism

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6
Q

original molecular cloning method for small scale investigations

A

PCR-restriction-ligation

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7
Q

other molecular cloning methods for larger scale investigations

A

ligation-free + recombination-based

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8
Q

applications of molecular cloning

A

Protein expression - prod of recombinant protein prods (e.g hormones - insulin + growth hormone)

Purification (recombinant vaccines + generation of genetically modified organisms (e.g knock-out mutants)

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9
Q

Molecular cloning experiments start with

A

integration of foreign DNA into suitable cloning vector

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10
Q

Factors effecting choice of vector

A

Size of foreign DNA to be cloned

Host organism that it will be maintained in (typically E.coli)

The final application (e.g protein expression + purification or construction of knock-out mutant)

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11
Q

what vector used if size of foreign DNA to be cloned is small

A

plasmid based cloning vector

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12
Q

plasmid based cloning vector consists of a small circular piece of DNA that contains:

A

A cloning site (where foreign DNA can be inserted)

A selectable marker - to allow for pos selection of transformants (e.g antibiotic resistance gene)

Elements necessary for propagation + maintenance in host organism (functional origin of replication ori is required for plasmid replication in E. coli)

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13
Q

Depending on intended application, cloning vectors can also contain

A

Elements necessary for expression (e.g promoter + ribosome binding site)

Reporter genes

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14
Q

function of PCR

A

Used in molecular cloning to amplify target DNA from chosen organism

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15
Q

PCR - If target DNA is prokaryotic

A

genomic DNA (gDNA) can be purified from cells + directly used as a template

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16
Q

what is PCR master mix

A

convenient ready-to-use formulation of PCR enzymes + reagents

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16
Q

PCR - If target DNA is eukaryotic

A

Not possible to amplify from gDNA due to presence of introns which must be removed prior to PCR amplification - how?

○ Purify RNA from host cells (as mRNAs will have undergone RNA splicing to remove introns)

○ Convert this to complementary DNA (cDNA) - using a retroviral enzyme, reverse transcriptase

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17
Q

3 PCR steps

A

Denaturation
Annealing (primer)
Elongation

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18
Q

denaturation step + temp

A

breaks complementary bonds between DNA frags
each dsDNA frag melts
yields 2 ssDNA frags

94-98C

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19
Q

annealing step + temp

A

temp dec to 48-68C

primer forms complementary bps w target seq

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20
Q

results of low annealing temp

A

primer binds imperfectly (similar but, not to identical seq in template)

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21
Q

result of high annealing temp

A

primer will not anneal to template

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22
Q

elongation step + temp

A

DNA polymerase synth new complementary DNA strands 5’ to 3’ direction

72C

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23
Q

function of agarose gel electrophoresis

A
  • Used to check purity + size of PCR prod by comparing w DNA ladder
  • DNA in gel stained w nucleic acid for visualisation (e.g SYBR Safe)
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24
Q

enzyme that generates sticky ends

A

restriction endonucleases
e.g EcoRI

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25
Q

enzyme that ligates sticky ends + PCR prod

A

T4 DNA ligase

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26
Q

do restriction endonucleases need to be the same + why/ not

A

no bc same sticky ends (of diff enzymes) can ligate together

27
Q

if single enzyme used to linearise vector its useful to

A

perform additional step prior to ligation - to prevent self-ligation of vector (i.e w/out insert)

28
Q

role of Antarctic phosphatase (AnP)

A

DNA modifying enzyme - dephosphorylates from 5’ and 3’ ends of DNA molecules

29
Q

2 main methods to transform e.coli

A

Chem transformation - E.coli treated w CaCl2 under cold conditions + then tempo heat shock- disrupts cell mem + allows exogenous DNA to enter cell

Electroporation - E.coli subjected to tempo electric field - punctures tiny holes in cell mem + allows exogenous DNA to enter cell

30
Q

next step after ligation

A

transformation - allows plasmid to be stably maintained + propagated for use in further experiments

31
Q

define tranformants

A

bacteria that has successfully taken up exogenous DNA from ligation reaction

32
Q

why do transformants need screening

A

can contain mixture of diff prods as well as desired cloned construct

identifies if colony carries correct plasmid

33
Q

transformant screening methods

A

Colony PCR - transformants lysed to release plasmid DNA by heating in water prior to PCR OR adding small amount of cells directly into PCR reaction (lysis occurs during denaturation step)

34
Q

next step after successfully cloning gene of interest into expression vector

A

transfer in a suitable host strain able to express the recombinant protein

35
Q

the promoter on the expression vector will only activate transcription in a particular
host, and the gene may either be

A

expressed at all times
OR
only expressed
under certain conditions (due to the presence of regulatory sequences in expression
vector)`

36
Q

a classical e.coli example of expression in recombinant proteins

A

T7 expression system

37
Q

how does T7 expression system work

A

utilises bacteriophage T7 promoter + several regulatory components of lac operon from E. coli

38
Q

When using t7 expression system, the cloned expression vector has to be transformed into a strain
of E. coli that produces bacteriophage T7 RNA polymerase + lac repressor, such as

A

BL21(DE3)

39
Q

how is expression of recombinant protein detected in host cell

A

Separate proteins based on electrophoretic mobility via polyacrylamide gel electrophoresis (PAGE) + then detect proteins using visible protein stain (Coomassie)/ Western blot

40
Q

PAGE can be performed using

A

whole cell lysates or w purified proteins

41
Q

how are proteins sep in native state

A

native PAGE
does not dentaure proteins

42
Q

how are proteins sep in denatured state

A

using sodium dodecyl sulphate = SDS-PAGE

43
Q

what does SDS-PAGE use + why

A

reducing agent (e.g BME/ DTT) to disrupt sulphide bonds + help fully denature the proteins

44
Q

N-POMC

A

76 AA peptide hormone which regulates size of adrenal

45
Q

why does N-POMC need to be cleaved at adrenal into smaller frags

A

to stim growth of gland

46
Q

what enzyme cleaves N-POMC into smaller frags

A

Trypsin-like serine protease, Adrenal Secretory Protease (AsP)

47
Q

SDS-PAGE seps proteins according to

A

physical size of polypep chain

48
Q

which size proteins move faster across gel

A

smaller

49
Q

how does coomassie staining work

A

coomassie brilliant blue dye binds to proteins based on electrostatic interactions

50
Q

how to protein bands become visible with coomassie staining

A

gel de-stained in water - improves contrast

51
Q

coomassie staining alternative

A

silver staining

52
Q

in western blotting after PAGE the polyacrylamide gel is placed onto a + why

A

transfer membrane (PVDF or
nitrocellulose)
which has a high affinity for proteins

53
Q

in western blotting why is electric field is applied across the electrodes

A

so that the proteins in the polyacrylamide gel migrate out of gel + bind to neg charged mem

ensures pattern
of proteins in gel is retained on mem

54
Q

why is blocking reagent used in western blotting (WB) + why important

A

protein in blocking reagent binds to regions
of mem that are not already bound by proteins (that were transferred from the gel
onto the membrane during the blotting step)

prevents non-specific
binding of detection Abs to the mem during next steps

55
Q

in WB, if the secondary Ab is conjugated to horseradish peroxidase (HRP), the
membrane is then treated with

A

enhanced chemiluminescent (ECL) Western blotting reagents
+ prod of light visualised using a digital imager

56
Q

what is used to detect signals from radioisotopes

A

autoradiography

57
Q

what is tween-20 + function

A

polysorbate surfactant

added to wash buffer to remove non
specifically bound + unbound Abs from mem

decr background signals when blot is dev + visualised

58
Q

hormones prod by adrenal medulla

A

adrenaline + noradrenaline

59
Q

hormones prod by adrenal cortex

A

aldosterone + cortisol
(steroid hormones)

60
Q

adrenal cortex reg by several pep hormones derived from

A

anterior pit

61
Q

why are His tags widely used in recombinant expression studies

A

can be detected w a commercial Ab

allows rapid
purification of the protein by affinity chromatography on an immobilized nickel column

62
Q

Ni co-ordinately binds to histidine residues but interaction can be reversed by

A

comp w imidazole
(chem which is structurally related to Histidine)

63
Q

why is vector pET32A chosen

A

contains elements
that enable high expression levels in E. coli

contains necessary seq to encode the His-tag

encodes a 109 residue thioredoxin tag which can
be expressed as an N-terminal fusion w gene of interest

64
Q

what is C-terminal HIS tagged fusion protein

A

A C-terminal HIS-tagged fusion protein refers to a recombinant protein that has been engineered to contain a short peptide sequence called a “histidine tag” at its C-terminus.

65
Q

what’s a multiple cloning site

A

short segment of DNA within a cloning vector that contains multiple unique restriction enzyme recognition sites - cleaved by restriction endonucleases

66
Q

start + stop codon

A

ATG

TAG

Molecular Genetics (1 decks)

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