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Biotechnological Gene Expression Flashcards

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1
Q

How do you make single copy plasmid yeast cells? Give name of yeast species also.

A

Saccharomyces cerevisiae

Use of the CEN gene
CEN = centromeric sequence (basically the centromere)

Allows plasmid to be inherited stably through mitosis (IPMAT)

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2
Q

Why is CEN good?

A

CEN = centromeric sequence

It is good because it is short (~300bp), which allows for it to be inserted into a yeast plasmid

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3
Q

What is auxotrophic complementation? With example(s)

A

Yeast plasmids containing recombinant gene cannot be selected for by antibiotics (like bacteria), so yeast vehicles are grown being deficient for an essential gene (e.g. HIS3), that produces and essential molecule (e.g. L-histidine). The recombinant plasmid contains the essential gene (e.g. HIS3), so only yeast containing the recombinant plasmid will survive.

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4
Q

How can auxotrophic complementation be used to increase plasmid copy number?

A

Species = Saccharomyces cerevisiae

Make Saccharomyces cells deficient of certain gene producing essential substance.

Make slightly defective gene that produces the essential substance an insert into recombinant plasmid (e.g. Leu2).

In order to get the required amount of essential substance, cells will select for producing the recombinant plasmid is a higher copy number.

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5
Q

How are plasmids in yeast made as high copy number of low copy number (with example(s))? Give yeast species in answer.

A

Saccharomyces cerevisiae

CEN = centromeric sequence
Treats the recombinant plasmid like an extra chromosome

2 micron (2u) sequence = 20-30 copies
is parasitic in origin
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6
Q

What kind of cellular function are strong promoters in yeast associated with? (example inc.)

A

Glycolytic reactions

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7
Q

What is a constitutive promoter?

A

Constant/consistent (on all the time) promoter, that is on all the time at a set rate, not inducible.

e.g. glycolytic promoters

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8
Q

How do you measure the strength of promoter expression?

Inc. examples and specific method

A

Use a reporter gene (e.g. LacZ or GFP (fluorescence) or Luciferase) and measure level of expression.

How separate? Cell cytometry

Cell-by-cell basis with GFP = fluorescence activated cell-sorting, small tube + laser excitation + reader

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9
Q

Give an example of a yeast origin of replication

A 
2 micron (multi-copy)
CEN (single copy)
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10
Q

Give an example of a yeast origin of replication

A

ARS - autonomous replication sequence

support efficient DNA replication initiation of extrachromosomal DNA

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11
Q

What is MCS?

A

mutliple cloning site

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12
Q

What is MCS?

A

multiple cloning site

  • is a DNA region within a Plasmid that contains multiple unique Restriction enzyme cut sites.

The multiple cloning site allows for foreign DNA to be inserted into the plasmid.

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13
Q

Example of an inducible promoter

A

e.g. Gal 1-10

Gal 1 and Gal 10 on opposite strands of the chromosome, divergent, different promoters.

BOTH
On in presence of galactose
Off in presence of glucose

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14
Q

What is good about T7 promoter gene?

A

Small gene

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15
Q

Why would you include a T7 promoter in a plasmid?

A

To promoter expression protein using T7 RNA polymerase.

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16
Q

What is AOX1?

A

Alcohol Oxidase 1
Pichia pastoris metabolises methanol not glucose.
methanol -> methanal uses AOX1

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17
Q

What is the value of Pichia pastoris?

A

can use methanol as carbon and energy source, so can use AOX1, which is much stronger than any promoter in S.cerevisiae.

Low manosylation (sugar decoration) of proteins 
-> low immunogenicity

Do not contain yeast origins of replication
Integrates into Pichia genome.

Can put in strong methanol solution, kills other microorganisms

Can grow at higher cell densities than S. cerevisiae

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18
Q

What is good about AOX1?

A

Promoter is very strong and inducible by methanol.

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19
Q

What is used to separate recombinant protein from tags? (with example)

A

Protease

TeV = Tobacco etch virus used to remove His6 tags by cutting linker.

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20
Q

Pichia pastoris promoters

A

FLD1 - methanol inducible

AOX1 - methanol inducible

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21
Q

Why chose to secrete proteins?

A

Ease of purification - 2K-3K other proteins in the cells when broken open (when cannot tag), if secrete, have a smaller pool

Need di-sulphide bonds (inside the cell is reducing and they cannot form there)

Tough cell walls - hard to break open, if secrete, don’t have to do this

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22
Q

What does penicillin act on?

A

Cell wall peptidogylcan synthesis

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23
Q

Why can antibiotic selection not work for yeast recombinant plasmid selection? (with example)

A

Yeast cells cannot be targeted by antibiotics.

e.g. penicillin target peptidoglycan synthesis (bacterial cell wall), which yeast does not req.

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24
Q

Metal ion affinity chromatography eluting agent for Ni sepharose column?

A

Imidazole

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25
How do you get a protein secreted in a yeast cell?
High-jacking of mating proteins (A and alpha) must be secreted from cells. Heterologous protein tagged onto this. pre (signal seq. cytoplasms to ER, then cut) /pro (ER to golgi, then cut) sequence. Then secreted from cell.
26
When does glycosylation occur during the secretion pathway?
Golgi apparatus
27
How is prokaryotic and eukaryotic translation distinct?
Eukaryotes - ribosomes bind at 5' end and scan along | Prokaryotes - ribosomes bind at the Shine-Dalgarno sequence
28
What is the Shine-Delgarno site?
Ribosome binding site on prokaryotic mRNA.
29
What is poly-cistronic mRNA?
AKA. operon Drive translation of 2 or more proteins (open reading frames) with a single promoter
30
What types of entry to the open reading frame do ribosomes use in prokaryotes?
Internal
31
What are the two subunits of prokaryotic ribosomes?
305 | 505
32
What is meant by terminator in the expression vector?
NOT the stop codon (translation) | Terminator = transcription terminator
33
Types of ORI
10-12 stringent control | 15-60 relaxed control
34
How is plasmid copy number controlled in bacteria?
Different origins of replication (ORIs)
35
Selectable markers for E.coli plasmids
tetR/bla/CmR
36
What is it and how does tetR work?
Tetracycline efflux pump Means of antibiotic resistance to tetracycline Pumps tetracycline out of the cell.
37
What is bla and how does it work?
Beta-lactamase Means of antibiotic resistance to ampicillin Cleaves ampicillin
38
What is CmR and how does it work?
Chloramphenicol acetyl transferase Means of antibiotic resistance Inactivates antibiotic with that group
39
Constitutive promoters in E.coli
``` Lac operon (switched on by IPTG) phoA (switched on by phosphate starvation) ```
40
Hybrid promoters in E.coli
tac, T7 based, but IPTG inducible | trc, (switched in by IPTG)
41
Types of transcription terminators
rho-independent, rRNA operon derived | rho-dependent
42
How is RNA pol. in bacteria different?
Does not scan in 5' UTR region (before open reading frame)
43
Why is it important to regulated mRNA turnover?
If rate is too fast, mRNA: - may be unstable
44
How can you regulate mRNA turnover (destruction)?
additions for artificial stabilisation (between promoter and ORF but before SD?) + some UTRs: add 5'UTR from ompA gene to the 5'UTR of an mRNA / HOW? blocks RNases at 5'UTR end +RNA stem loop (self-complimentary RNA) / HOW? blocks RNases at 5'UTR end
45
Why might proteins be turned over?
incorrect folding
46
How can you minimise protein turnover?
Limiting as extruded from ribosome - chaperone proteins -> better folding e.g. GroESL
47
Example of a chaperone protein
GroESL
48
How can the formation of di-sulphide bonds be increased with heterologous protein expression in bacteria?
Increased formation of DSBA/DSPB with the protein. DSBA - disulphide bond forming enzyme periplasm of Gram negative bacteria catalyses formation of disulphide bridges
49
Why is temperature important for expressed protein solubility?
protein solubility can be degraded at high levels of expression (& higher temperature) why? - insufficient time for protein folding
50
Affinity tags
glutathione transferase (GST) his6 tags maltose binding protein
51
Affinity chromatography example (non-metal)
glutathione transferase (GST) tags stick to glutathione column, glutathione (soluble form) used to elute maltose binding protein tags stick to maltose column, eluted with maltose (soluble form).
52
What is special about imidazole?
has histidine side chain, lone pair of nitrogens co-ordinate with nickel ions on the column
53
How does affinity chromatography elution work?
Wash column through with soluble form of something that binds to the ligand (column). Ligand would bind the soluble form in preference to the immobilised form.
54
Why tag with ligands?
Improve solubility (e.g. maltose binding protein) Improve stability Purification
55
Where are disulphide bridges formed in proteins in bacteria?
Periplasmic Space
56
What sort of environment is required for the formation of disulphide bonds?
Oxidising
57
Where are disulphide bridges formed in proteins in eukaryotic cells?
Endoplasmic Reticulum
58
How are proteins moved to the periplasmic space from the cytoplasm?
signal sequence on n-terminus of the protein to direct it to a secretory pathway fusion partners for co-secretion tack the protein onto a known secreted molecule
59
What makes disulphide bonds in proteins in eukaryotic cells?
Protein Disulphide Isomerase
60
General rule with E.coli codon bias
A-T biased | favour U ending codon over C ending codon
61
codon adaptive score
codon bias measurement
62
Argument against the codon bias factor in heterologous gene expression
mRNA secondary structures seem to be more predictive
63
Selectivity of codon bias effect
For a number of genes - within the first 28 codons of a protein codon bias seemed to have a bigger impact Codon bias can be rescued by adding first 28 codons efficiently expressed DO NOT KNOW WHY
64
Insect cells recombinant protein expression example
Baculovirus infects insect cells - bacmid plasmids can be used to produce recombinant proteins from insect cells. Baculovirus polyhedrin promoter - promoter for capsid formation, is very powerful drives expression at high level in insect cells
65
What is a bacmid?
Baculovirus plasmid (recombinant) Used to infect infect cells and cause viral production of a recombinant protein.
66
Origin of the T7 promoter
phage
67
What is the T7 lysozyme system?
Targets T7 polymerase for degradation.
68
What is the purpose of the T7 lysozyme system?
To stop background low levels of T7 polymerase from working from leaky expression from T7 control by IPTG.
69
What happens to T7 lysozyme system under IPTG induction of T7 polymerase?
T7 polymerase production overwhelms the T7 lysozyme system.
70
How to control IPTG induction levels in T7 polymerase system?
lacY transporter determines how much IPTG enters the cell
71
Combatting codon bias in E.coli
Re-engineering of codon sequence Add "rare" codon tRNA genes to the recombinant plasmid, to be expressed with the recombinant protein of interest. e.g. Rosetta strain
72
Novel way to help folding of recombinant proteins
Origami strains of E.coli | Make the cytoplasm less reducing, enabling disulphide bond formation
73
Example of an antibody protein tag
FLAG
74
Key features of insulin expression
Requires secretion Has disulphide bonds Is secreted in sections
75
Example of a synthetic promoter/transcription factor
TALEs
76
What is a TALE?
transcription activator like effector protein 33-35 amino repeats
77
What makes up a TALE?
N- terminal = protein secretion and translocation signals Central tandem repeats domain, each repeat(amino acids 12 and 13) matter for the base that it targets (ATCG) on RNA pol. II C-terminal = gets protein into the nucleus and transcription activation domain
78
How to check have made a protein and that it binds properly (with example)? (Eurkaryotes)
TALEs TALE = promoter/transcription factor Tag TALE protein with mCherry = red Make it so complex(TALE+RNApol.II) = cyan fluorescing
79
What is ortholonal in the context of TALEs?
Isolated from host biochemistry | - will only bind to what designed to bind to
80
Humanizing proteins through glycosylation patterns (species specific)
Knock out yeast glycosylation genes, replace with human glycosylation genes Replacing Pichia pastoris glycosylation enzymes with human glycosylation enzymes in the Golgi apparatus
81
Case study for S. cerevisiae and human therapeutics
Hep B vaccine
82
How Hep B vaccine made?
Recombinant plasmid Yeast cracked open to retrieve viral protein Assembles as viral capsid in cytoplasm Separate virus-like particles by size
83
Control of translation initiation in prokaryotes using the Shine-Delgarno region
Repression by trans-acting proteins - masking the Shine-Delgarno seq. Modulation of secondary structure of ribosome binding site Altering sequence of Shine-Delagarno seq.
84
Things to avoid when optimizing maximal flow-through in gene expression
Not balancing the enzymes in each step of pathway -> Protein burden Intermediate accumulation Low flux
85
How to optimize maximal flow-through in gene expression
one promoter - operon | diff. ribosome binding seq. to balance expression
86
Example of how test to optimize maximal flow-through in gene expression
RBS binding site selection ``` Different coloured (fluorescence) marker genes for each protein -> distinct end colour depending upon combined expression success of the proteins ``` e.g. strong/weak/strong strong/strong/strong e.g. cyan, mCHerry and GFP
87
Translational coupling (with example) Prokaryotes
Ribosomes are passed on to the next open reading frame, so the efficiency of the previous gene affects the efficiency of the next gene. EXPERIMENT e.g. strong/weak/strong strong/strong/strong e.g. cyan, mCHerry and GFP
88
How is mRNA expression primarily regulated?
the ribosome binding site affinity NOT codon bias
89
Limiting points of transcription
initiation | elongation - codon bias

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