QBM exam | Questions Solved with
Verified Correct Answers | Latest Update
2024-2025
Mammalian cells as protein expressors - ANSWER - Take large amounts of animal organs
and purify proteins directly
- Slow - hard to keep alive
- Expensive with
- Low yield
- WIll get final protein with proper modifications - that is, in its *native state*
Bacteria are poor compared to mammalian cells when it comes to glycosylation/folding
of expressed proteins - ANSWER True
Insulin expression in prokaryotic vs eukaryotic systems - ANSWER E. coli - requires expression,
lysis, purificaiton, and refolding to get it
S. cervisiae - expresses straight into supernatent
What makes E. coli so widely used in a lab? - ANSWER - Takes up foreign DNA easily
- Grows fast, easy to regualte
- *Lacks most proteases*
,- lack recombination
What are some example E. coli cell line strains? - ANSWER 1) *BL21* = contain T7
RNA polymerase gene
2) *B834* = methionine auxotrophs
3) pLysS = contains plasmid pLysS that expresses T7 lysozyme to inhibit basal levels
of expression (toxic compounds)
4) Tuner - lac permease mutation allows uniform entry of IPTG into cells - allows to control
induction level - "tighter control"
5) Origami - thioredoxin reductase, glutathione reductase mtuations to *enhance doulbe bond
formation*
6) Mach1 = fast cell growth
Actual protein production method from bacteria? - ANSWER 1) Vector of interest clone is
grown at 37C in LB (lysogeny media - typrtone, yeast extract, NaCl,) overnight with ampicillin
to select for bacterias not carrying plasmid - requries addition of fresh ampicillin
2) Grow to an *optical density of 600nm at 0.6* (exponential growth phase)
3) Grows mL to L of bacteria
4) Induce with IPTG
5) Spin down cells, lyse - get 1mg-500mg for every L of bacteria
IPTG - ANSWER Isopropyl-beta-D-thiogalactopyranoside
A lactose "variant" that *cannot be metabolized* that will displace the lac reporessor from
the lac operator (allowing T7 operator to be activated)
IPTG induction is reversible - ANSWER False; it is "permanent" on mode,b ecause it cannot be
metabolized
,We induce (add IPTG) when... - ANSWER When the OD at 600nm is approixmately 0.2 to 0.6
(bacteria is at its exponential growth phase)
(This can be visualized)
That is, get protein during the cell's maximal level of competance
Lac operon is normally used by bacteria to... In the presnec of lactose, this operon is - ANSWER
Express the beta-galactosidase, permease, and transacetylase proteins for lactose breakdown
Lactose will bind to the *lac repressor* and inactivate it via inducing - allows transcription
LacI - ANSWER On the lac operon will make lac inhibitor/repressor that binds to its
own operator to repress transcription
Bacteriophage T7 will bring... - ANSWER Infects E. coli
Its own RNA polymerase genes, so if it gets transcribed then the T7 RNA polymerase will
be made and will bind to its own T7 promoter sequence
pGEX vector is an example of what system - ANSWER a Ptac vector system
pTac vector system - ANSWER - Has a Ptac promoter that is silenced by lac repressor protein
- Gene of interest downstream of promoter (like GST)
- System is turned on by IPTG, removes the lac repressor
- *E. col's RNA polymerase will transcribe the gene of interest, and E. coli's ribosomes
willl synthesize the protein of interest*
pTac uses a T7 promtoer - ANSWER False, an E. coli promoter
, Disadvantage of a pTac system - ANSWER "LEaky" - background expression even in the "off site"
This uncontrolled expression may lead to misfolded protein, toxicity if applicable
pET system - ANSWER Phage Vector System
- Uses host proteins, bacteriphoage proteins, and lac operon, ampicillin resitance
- Some strains of E. coli (BL21) contain *bacteriophage T7 RNA polymerase* with lac operon
upstream
- Lac operon bound to repressor, until IPTG turns it on
- E. coli's RNAP and ribosomes transcribe/transalte the T7 RNA polymerase on *host genome*
- T7 RNA polymerase targets the *T7 promoter* on the plasmid and transcribes our gene
of interest
- mRNA with gene of interest translated by *E. coli's ribosome*
pET advantages - ANSWER Tighter control - less background expression
- High expression of a *recombinant protein*
How can the pET system be further "in check" - ANSWER T7 lysozyme can inactivate expression
of T7 RNA polymerase binding to its promoter if we need to control background "leakiness"
You can use a Ptac plasmid for BL21 - ANSWER True (even though BL21 has T7 RNAP)
How can we extract the protein from the cells after inducing expression? - ANSWER - Pellet cells
in centrifuge, discard media
- Lysate/"homogenate" in a buffer which the protein is stable
Verified Correct Answers | Latest Update
2024-2025
Mammalian cells as protein expressors - ANSWER - Take large amounts of animal organs
and purify proteins directly
- Slow - hard to keep alive
- Expensive with
- Low yield
- WIll get final protein with proper modifications - that is, in its *native state*
Bacteria are poor compared to mammalian cells when it comes to glycosylation/folding
of expressed proteins - ANSWER True
Insulin expression in prokaryotic vs eukaryotic systems - ANSWER E. coli - requires expression,
lysis, purificaiton, and refolding to get it
S. cervisiae - expresses straight into supernatent
What makes E. coli so widely used in a lab? - ANSWER - Takes up foreign DNA easily
- Grows fast, easy to regualte
- *Lacks most proteases*
,- lack recombination
What are some example E. coli cell line strains? - ANSWER 1) *BL21* = contain T7
RNA polymerase gene
2) *B834* = methionine auxotrophs
3) pLysS = contains plasmid pLysS that expresses T7 lysozyme to inhibit basal levels
of expression (toxic compounds)
4) Tuner - lac permease mutation allows uniform entry of IPTG into cells - allows to control
induction level - "tighter control"
5) Origami - thioredoxin reductase, glutathione reductase mtuations to *enhance doulbe bond
formation*
6) Mach1 = fast cell growth
Actual protein production method from bacteria? - ANSWER 1) Vector of interest clone is
grown at 37C in LB (lysogeny media - typrtone, yeast extract, NaCl,) overnight with ampicillin
to select for bacterias not carrying plasmid - requries addition of fresh ampicillin
2) Grow to an *optical density of 600nm at 0.6* (exponential growth phase)
3) Grows mL to L of bacteria
4) Induce with IPTG
5) Spin down cells, lyse - get 1mg-500mg for every L of bacteria
IPTG - ANSWER Isopropyl-beta-D-thiogalactopyranoside
A lactose "variant" that *cannot be metabolized* that will displace the lac reporessor from
the lac operator (allowing T7 operator to be activated)
IPTG induction is reversible - ANSWER False; it is "permanent" on mode,b ecause it cannot be
metabolized
,We induce (add IPTG) when... - ANSWER When the OD at 600nm is approixmately 0.2 to 0.6
(bacteria is at its exponential growth phase)
(This can be visualized)
That is, get protein during the cell's maximal level of competance
Lac operon is normally used by bacteria to... In the presnec of lactose, this operon is - ANSWER
Express the beta-galactosidase, permease, and transacetylase proteins for lactose breakdown
Lactose will bind to the *lac repressor* and inactivate it via inducing - allows transcription
LacI - ANSWER On the lac operon will make lac inhibitor/repressor that binds to its
own operator to repress transcription
Bacteriophage T7 will bring... - ANSWER Infects E. coli
Its own RNA polymerase genes, so if it gets transcribed then the T7 RNA polymerase will
be made and will bind to its own T7 promoter sequence
pGEX vector is an example of what system - ANSWER a Ptac vector system
pTac vector system - ANSWER - Has a Ptac promoter that is silenced by lac repressor protein
- Gene of interest downstream of promoter (like GST)
- System is turned on by IPTG, removes the lac repressor
- *E. col's RNA polymerase will transcribe the gene of interest, and E. coli's ribosomes
willl synthesize the protein of interest*
pTac uses a T7 promtoer - ANSWER False, an E. coli promoter
, Disadvantage of a pTac system - ANSWER "LEaky" - background expression even in the "off site"
This uncontrolled expression may lead to misfolded protein, toxicity if applicable
pET system - ANSWER Phage Vector System
- Uses host proteins, bacteriphoage proteins, and lac operon, ampicillin resitance
- Some strains of E. coli (BL21) contain *bacteriophage T7 RNA polymerase* with lac operon
upstream
- Lac operon bound to repressor, until IPTG turns it on
- E. coli's RNAP and ribosomes transcribe/transalte the T7 RNA polymerase on *host genome*
- T7 RNA polymerase targets the *T7 promoter* on the plasmid and transcribes our gene
of interest
- mRNA with gene of interest translated by *E. coli's ribosome*
pET advantages - ANSWER Tighter control - less background expression
- High expression of a *recombinant protein*
How can the pET system be further "in check" - ANSWER T7 lysozyme can inactivate expression
of T7 RNA polymerase binding to its promoter if we need to control background "leakiness"
You can use a Ptac plasmid for BL21 - ANSWER True (even though BL21 has T7 RNAP)
How can we extract the protein from the cells after inducing expression? - ANSWER - Pellet cells
in centrifuge, discard media
- Lysate/"homogenate" in a buffer which the protein is stable
