This summary consists of 26 pages. It is a summary of the techniques lectures given in the 2nd term of the 2nd year. The summary contains clear figures and assignments (with answers) given in class
BL21(DE3)pLysS competent cells
BL21: strain of E. coli
DE3: a gene that’s in the BL21 genome. It encodes for
a promoter, lac operator and the T7 RNA polymerase
pLysS: extra plasmid present (pLys)
Plasmids used in the lab
Plasmid name Protein tag Protein purification Promoter Protein Antibiotic
method expression resistance
inducer
pBAD/His A N-His Affinity chromatography; Ara BAD Arabinose Ampicillin (bla
Ni2+ column promoter gene)
pGEX 4-T-1 N-GST Affinity chromatography; Tac promoter IPTG Ampicillin (bla
glutathione column gene)
pET 15 N-His Affinity chromatography; T7 promoter IPTG Ampicillin (bla
Ni2+ column gene)
L-arabinose induction
araC protein is made and will bind to the ara operator and 1 of
the ara inducers → conformation will be formed→ pBAD is blocked
and RNA polymerase cannot bind → operon is repressed
When arabinose is present: arabinose binds to the araC
protein → conformational change → araC binds to both
inducers (araI1 and araI2) → pBAD will be functional; RNA
polymerase can bind to it → GOI can be transcribed
pBAD/His
Regulatory sequence: araC → inducer molecule: arabinose
The absence of L-arabinose produces very low levels or transcription
from pBAD
In the presence of L-arabinose, expression from pBAD is turned on
BL21(DE3)pLysS + pBAD/His
RNA polymerase will (always) be transcribed → RNA polymerase
will bind to the promoter of araC and the araC protein will be
made. AraC protein will bind to the ara operator and ara inducer
sites → thereby blocking the pBAD promoter → GOI cannot be
made
Arabinose induction BL21(DE3)pLysS + pBAD/His
RNA polymerase will bind to the promoter of araC and the araC
protein will be made. Arabinose can bind to this protein and some
conformational change will happen. AraC will bind to ara inducers
→ pBAD not inhibited → another RNA polymerase binds to pBAD →
transcription of GOI
1
,IPTG induction
LacI codes for the lac repressor proteins
Lac repressor proteins can bind to the lac operator →
transcription/promoter blocked → GOI cannot be transcribed
When IPTG is present: IPTG binds to the repressor → repressors are
inhibited. Repressor cannot bind to the lac operator no more →
promoter is not blocked → transcription can occur
pGEX-4T-1
Regulatory sequence: lacI with the lac operator
Inducer molecule: IPTG
Expression is under the control of the tac promoter, which
is induced by the lactose isopropyl β-D-thiogalatoside (IPTG)
GST occurs naturally as a 26 kDa protein that can be
expressed in E. coli
BL21(DE3)pLysS + pGEX 4T1
Without IPTG → still expression of RNA polymerase → bind to the
promoter of lacI → lacI protein will be made → repressor protein will
bind to the operator → promoter is blocked → no transcription of
GOI
IPTG induction BL21(DE3)pLysS + pGEX 4T1
RNA polymerase binds to the promoter of lacI → lacI repressor
protein will be made → IPTG binds to respressors and take them
away → repressor cannot bind to the lacI operator → RNA
polymerase can bind to promoter → transcription of GOI
BL21(DE3)pLysS + pET 15
Without IPTG → RNA polymerase made → binds to promoter of lacI
→ lacI repressor protein are made → repressor bind to lacO
operator → T7 promoter will be blocked → no transcription of GOI
IPTG induction BL21(DE3)pLysS + pET 15
RNA polymerase made → binds to promoter of lacI → lacI repressor
protein are made → IPTG blocks repressor protein → repressor
protein cannot bind to lacI operator
The genome of BL21 also contains a lacI gene, so lacI repressor
proteins are also made over there → IPTG binds to repressor.
Therefore, RNA polymerase will bind to promoter for T7 RNA polymerase
T7 RNA polymerase is made → binds to T7 promoter of plasmid → transcription of GOI
2
, pLysS
Feature Stands for Function in plasmid
CmR Chloramphenicol resistance BL21(DE3)pLys selection. Cells
gene will not lose pLys when
culturing with chloramphenicol
p15A ori Origin of replication BL21(DE3) cells will also
initiation. Adapted from replicate pLys when they divide
plasmid 15A, so bacterial
T7 Lysozyme that inhibits T7 Reduction of basal T7 driven
lysozyme RNA polymerase expression of inserted gene
No MCS Multiple cloning site This plasmid is not meant for
cloning or expression of a GOI
pET-15b and BL21(DE3)pLysS
To prevent expression of IDH1 by leaky expression of T7 RNA
polymerase: T7 lysozyme inactivates T7 RNA polymerase. Because is
this, the T7 lysozyme is still present, but there is an overexpression
of the T7 RNA polymerase, and that can activate the GOI
Expression of eukaryotic protein in bacteria
Bacterial expression (E. coli) is the most common expression system employed for the
production of recombinant proteins
Advantages
1. Easy to manipulate
2. Inexpensive in culturing
3. Fast of generation of recombinant protein
1. Disadvantage: codon bias
Inefficient expression due to codon usage bias:
Codon usage bias is the tendency of some organisms to use certain DNA triplets in
preference to other (equally capable) triplets in building amino acids.
So, when looking at this table, you can see that every organism can “choose”
6 different codons to code for arginine. Though, organisms have a preference
and use one codon more than the other, because not every codon is
translated in the most efficient way in each organism.
2. Disadvantage: misfolding proteins
Higher risk of misfolding proteins due to heterogeneous expression; misfolded
proteins accumulate in inclusion bodies (aggregation of lots of misfolded
proteins), and will eventually be in the cell pellet when isolating protein of
interest
3
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