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Biotechnology > Biotechnological Gene Expression > Flashcards

Biotechnological Gene Expression Flashcards

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
Q

How do you get a protein secreted in a yeast cell?

A

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.

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26
Q

When does glycosylation occur during the secretion pathway?

A

Golgi apparatus

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27
Q

How is prokaryotic and eukaryotic translation distinct?

A

Eukaryotes - ribosomes bind at 5’ end and scan along

Prokaryotes - ribosomes bind at the Shine-Dalgarno sequence

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28
Q

What is the Shine-Delgarno site?

A

Ribosome binding site on prokaryotic mRNA.

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29
Q

What is poly-cistronic mRNA?

A

AKA. operon

Drive translation of 2 or more proteins (open reading frames) with a single promoter

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30
Q

What types of entry to the open reading frame do ribosomes use in prokaryotes?

A

Internal

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31
Q

What are the two subunits of prokaryotic ribosomes?

A

305

505

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32
Q

What is meant by terminator in the expression vector?

A

NOT the stop codon (translation)

Terminator = transcription terminator

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33
Q

Types of ORI

A

10-12 stringent control

15-60 relaxed control

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34
Q

How is plasmid copy number controlled in bacteria?

A

Different origins of replication (ORIs)

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35
Q

Selectable markers for E.coli plasmids

A

tetR/bla/CmR

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36
Q

What is it and how does tetR work?

A

Tetracycline efflux pump
Means of antibiotic resistance to tetracycline
Pumps tetracycline out of the cell.

37
Q

What is bla and how does it work?

A

Beta-lactamase
Means of antibiotic resistance to ampicillin
Cleaves ampicillin

38
Q

What is CmR and how does it work?

A

Chloramphenicol acetyl transferase
Means of antibiotic resistance
Inactivates antibiotic with that group

39
Q

Constitutive promoters in E.coli

A 
Lac operon (switched on by IPTG)
phoA (switched on by phosphate starvation)
40
Q

Hybrid promoters in E.coli

A

tac, T7 based, but IPTG inducible

trc, (switched in by IPTG)

41
Q

Types of transcription terminators

A

rho-independent, rRNA operon derived

rho-dependent

42
Q

How is RNA pol. in bacteria different?

A

Does not scan in 5’ UTR region (before open reading frame)

43
Q

Why is it important to regulated mRNA turnover?

A

If rate is too fast,
mRNA:
- may be unstable

44
Q

How can you regulate mRNA turnover (destruction)?

A

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
Q

Why might proteins be turned over?

A

incorrect folding

46
Q

How can you minimise protein turnover?

A

Limiting as extruded from ribosome
- chaperone proteins -> better folding

e.g. GroESL

47
Q

Example of a chaperone protein

A

GroESL

48
Q

How can the formation of di-sulphide bonds be increased with heterologous protein expression in bacteria?

A

Increased formation of DSBA/DSPB with the protein.

DSBA - disulphide bond forming enzyme
periplasm of Gram negative bacteria
catalyses formation of disulphide bridges

49
Q

Why is temperature important for expressed protein solubility?

A

protein solubility can be degraded at high levels of expression (& higher temperature)

why? - insufficient time for protein folding

50
Q

Affinity tags

A

glutathione transferase (GST)
his6 tags
maltose binding protein

51
Q

Affinity chromatography example (non-metal)

A

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
Q

What is special about imidazole?

A

has histidine side chain, lone pair of nitrogens co-ordinate with nickel ions on the column

53
Q

How does affinity chromatography elution work?

A

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
Q

Why tag with ligands?

A

Improve solubility (e.g. maltose binding protein)
Improve stability
Purification

55
Q

Where are disulphide bridges formed in proteins in bacteria?

A

Periplasmic Space

56
Q

What sort of environment is required for the formation of disulphide bonds?

A

Oxidising

57
Q

Where are disulphide bridges formed in proteins in eukaryotic cells?

A

Endoplasmic Reticulum

58
Q

How are proteins moved to the periplasmic space from the cytoplasm?

A

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
Q

What makes disulphide bonds in proteins in eukaryotic cells?

A

Protein Disulphide Isomerase

60
Q

General rule with E.coli codon bias

A

A-T biased

favour U ending codon over C ending codon

61
Q

codon adaptive score

A

codon bias measurement

62
Q

Argument against the codon bias factor in heterologous gene expression

A

mRNA secondary structures seem to be more predictive

63
Q

Selectivity of codon bias effect

A

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
Q

Insect cells recombinant protein expression example

A

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
Q

What is a bacmid?

A

Baculovirus plasmid (recombinant)

Used to infect infect cells and cause viral production of a recombinant protein.

66
Q

Origin of the T7 promoter

A

phage

67
Q

What is the T7 lysozyme system?

A

Targets T7 polymerase for degradation.

68
Q

What is the purpose of the T7 lysozyme system?

A

To stop background low levels of T7 polymerase from working from leaky expression from T7 control by IPTG.

69
Q

What happens to T7 lysozyme system under IPTG induction of T7 polymerase?

A

T7 polymerase production overwhelms the T7 lysozyme system.

70
Q

How to control IPTG induction levels in T7 polymerase system?

A

lacY transporter determines how much IPTG enters the cell

71
Q

Combatting codon bias in E.coli

A

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
Q

Novel way to help folding of recombinant proteins

A

Origami strains of E.coli

Make the cytoplasm less reducing, enabling disulphide bond formation

73
Q

Example of an antibody protein tag

A

FLAG

74
Q

Key features of insulin expression

A

Requires secretion
Has disulphide bonds
Is secreted in sections

75
Q

Example of a synthetic promoter/transcription factor

A

TALEs

76
Q

What is a TALE?

A

transcription activator like effector protein

33-35 amino repeats

77
Q

What makes up a TALE?

A

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
Q

How to check have made a protein and that it binds properly (with example)?

(Eurkaryotes)

A

TALEs
TALE = promoter/transcription factor

Tag TALE protein with mCherry = red

Make it so complex(TALE+RNApol.II) = cyan fluorescing

79
Q

What is ortholonal in the context of TALEs?

A

Isolated from host biochemistry

- will only bind to what designed to bind to

80
Q

Humanizing proteins through glycosylation patterns (species specific)

A

Knock out yeast glycosylation genes, replace with human glycosylation genes

Replacing Pichia pastoris glycosylation enzymes with human glycosylation enzymes in the Golgi apparatus

81
Q

Case study for S. cerevisiae and human therapeutics

A

Hep B vaccine

82
Q

How Hep B vaccine made?

A

Recombinant plasmid
Yeast cracked open to retrieve viral protein
Assembles as viral capsid in cytoplasm
Separate virus-like particles by size

83
Q

Control of translation initiation in prokaryotes using the Shine-Delgarno region

A

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
Q

Things to avoid when optimizing maximal flow-through in gene expression

A

Not balancing the enzymes in each step of pathway ->

Protein burden
Intermediate accumulation
Low flux

85
Q

How to optimize maximal flow-through in gene expression

A

one promoter - operon

diff. ribosome binding seq. to balance expression

86
Q

Example of how test to optimize maximal flow-through in gene expression

A

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
Q

Translational coupling (with example)

Prokaryotes

A

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
Q

How is mRNA expression primarily regulated?

A

the ribosome binding site affinity NOT codon bias

89
Q

Limiting points of transcription

A

initiation

elongation - codon bias

Biotechnology (5 decks)

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