Lab Practical Flashcards
purpose of vector
used to carry foreign DNA fragments into host cells
role of promoters
reg gene expression
markers
aid in selection of transformed cells
tags
facilitate protein purification/ detection
what is molecular cloning
set of experimental methods used to construct recombinant DNA molecules that can be replicated in suitable host organism
original molecular cloning method for small scale investigations
PCR-restriction-ligation
other molecular cloning methods for larger scale investigations
ligation-free + recombination-based
applications of molecular cloning
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)
Molecular cloning experiments start with
integration of foreign DNA into suitable cloning vector
Factors effecting choice of vector
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)
what vector used if size of foreign DNA to be cloned is small
plasmid based cloning vector
plasmid based cloning vector consists of a small circular piece of DNA that contains:
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)
Depending on intended application, cloning vectors can also contain
Elements necessary for expression (e.g promoter + ribosome binding site)
Reporter genes
function of PCR
Used in molecular cloning to amplify target DNA from chosen organism
PCR - If target DNA is prokaryotic
genomic DNA (gDNA) can be purified from cells + directly used as a template
what is PCR master mix
convenient ready-to-use formulation of PCR enzymes + reagents
PCR - If target DNA is eukaryotic
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
3 PCR steps
Denaturation
Annealing (primer)
Elongation
denaturation step + temp
breaks complementary bonds between DNA frags
each dsDNA frag melts
yields 2 ssDNA frags
94-98C
annealing step + temp
temp dec to 48-68C
primer forms complementary bps w target seq
results of low annealing temp
primer binds imperfectly (similar but, not to identical seq in template)
result of high annealing temp
primer will not anneal to template
elongation step + temp
DNA polymerase synth new complementary DNA strands 5’ to 3’ direction
72C
function of agarose gel electrophoresis
- 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)
enzyme that generates sticky ends
restriction endonucleases
e.g EcoRI
enzyme that ligates sticky ends + PCR prod
T4 DNA ligase
do restriction endonucleases need to be the same + why/ not
no bc same sticky ends (of diff enzymes) can ligate together
if single enzyme used to linearise vector its useful to
perform additional step prior to ligation - to prevent self-ligation of vector (i.e w/out insert)
role of Antarctic phosphatase (AnP)
DNA modifying enzyme - dephosphorylates from 5’ and 3’ ends of DNA molecules
2 main methods to transform e.coli
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
next step after ligation
transformation - allows plasmid to be stably maintained + propagated for use in further experiments
define tranformants
bacteria that has successfully taken up exogenous DNA from ligation reaction
why do transformants need screening
can contain mixture of diff prods as well as desired cloned construct
identifies if colony carries correct plasmid
transformant screening methods
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)
next step after successfully cloning gene of interest into expression vector
transfer in a suitable host strain able to express the recombinant protein
the promoter on the expression vector will only activate transcription in a particular
host, and the gene may either be
expressed at all times
OR
only expressed
under certain conditions (due to the presence of regulatory sequences in expression
vector)`
a classical e.coli example of expression in recombinant proteins
T7 expression system
how does T7 expression system work
utilises bacteriophage T7 promoter + several regulatory components of lac operon from E. coli
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
BL21(DE3)
how is expression of recombinant protein detected in host cell
Separate proteins based on electrophoretic mobility via polyacrylamide gel electrophoresis (PAGE) + then detect proteins using visible protein stain (Coomassie)/ Western blot
PAGE can be performed using
whole cell lysates or w purified proteins
how are proteins sep in native state
native PAGE
does not dentaure proteins
how are proteins sep in denatured state
using sodium dodecyl sulphate = SDS-PAGE
what does SDS-PAGE use + why
reducing agent (e.g BME/ DTT) to disrupt sulphide bonds + help fully denature the proteins
N-POMC
76 AA peptide hormone which regulates size of adrenal
why does N-POMC need to be cleaved at adrenal into smaller frags
to stim growth of gland
what enzyme cleaves N-POMC into smaller frags
Trypsin-like serine protease, Adrenal Secretory Protease (AsP)
SDS-PAGE seps proteins according to
physical size of polypep chain
which size proteins move faster across gel
smaller
how does coomassie staining work
coomassie brilliant blue dye binds to proteins based on electrostatic interactions
how to protein bands become visible with coomassie staining
gel de-stained in water - improves contrast
coomassie staining alternative
silver staining
in western blotting after PAGE the polyacrylamide gel is placed onto a + why
transfer membrane (PVDF or
nitrocellulose)
which has a high affinity for proteins
in western blotting why is electric field is applied across the electrodes
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
why is blocking reagent used in western blotting (WB) + why important
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
in WB, if the secondary Ab is conjugated to horseradish peroxidase (HRP), the
membrane is then treated with
enhanced chemiluminescent (ECL) Western blotting reagents
+ prod of light visualised using a digital imager
what is used to detect signals from radioisotopes
autoradiography
what is tween-20 + function
polysorbate surfactant
added to wash buffer to remove non
specifically bound + unbound Abs from mem
decr background signals when blot is dev + visualised
hormones prod by adrenal medulla
adrenaline + noradrenaline
hormones prod by adrenal cortex
aldosterone + cortisol
(steroid hormones)
adrenal cortex reg by several pep hormones derived from
anterior pit
why are His tags widely used in recombinant expression studies
can be detected w a commercial Ab
allows rapid
purification of the protein by affinity chromatography on an immobilized nickel column
Ni co-ordinately binds to histidine residues but interaction can be reversed by
comp w imidazole
(chem which is structurally related to Histidine)
why is vector pET32A chosen
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
what is C-terminal HIS tagged fusion protein
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.
what’s a multiple cloning site
short segment of DNA within a cloning vector that contains multiple unique restriction enzyme recognition sites - cleaved by restriction endonucleases
start + stop codon
ATG
TAG
