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Genes to Proteins > Genetic Engineering and Protein Engineering - Genes > Flashcards

Genetic Engineering and Protein Engineering - Genes Flashcards

1
Q

What is a lipase?

A

Cleaves ester bonds, using water, in triacylglycerols to produce glycerol and fatty acids

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2
Q

What residue is present in lipase that is important in the break down of an ester bond?

A

Serine

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3
Q

Why doesnt serine attack the ester bond by itself in lipase, what does it need to do this?

A

It is not nucleophilic enough - it is activated by histidine

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4
Q

What are the 4 steps involved during the breakdown an ester bond by lipase?

A

1) Attack of ester by serine after activation by histidine - formation of oxyanion
2) Ester bond reforms and alcohol is lost
3) Water is deprotonated by histidine and attack ester once more - formation of 2nd oxyanion
4) C=O reforms - carboxylic acid is lost from serine and serine attacks protonated histidine to reform starting structures for lipase residues

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5
Q

What is kinetic resolution in respect to lipase in water?

A

Lipase only reactions with a specific chiral ester to form one enantiomerically pure product and unreacted SM of the other enantiomer

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6
Q

What occurs when a racemic alcohol is mixed with anhydrous organic solvent and lipase?

A

Enantioselective acylation occurs

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7
Q

Where does the gene sequence of lipase come from and what are the problems with it?

A

Rhizomucor miehei - a fungus. It is eukaryotic which raises issues

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8
Q

What is RML?

A

Rhizomuco miehei lipase - the lipase we want

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9
Q

What are the two ways in which the RML gene could be obtained and which is preferred?

A

1) Obtain the genomic DNA from the fungus itself
2) Have the gene synthesised by a company - preferred

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10
Q

What is an intron?

A

A section of DNA that is not coded into the protein. Must be spliced (removed) from mRNA - not present in prokaryotic mRNA

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11
Q

What needs to happen to membrane anchors, signal sequences and propeptides and what do they all do?

A

Useless bits of protein that need to be removed
Membrane anchor - attaches protein to cell membrane
Signal sequences - tells a cell where to send a protein
Propeptides - cleaved to produce a mature protein

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12
Q

What is codon optimisation and why is it needed for gene design?

A

Codon expression is dependant on organism - Leucine has six possible codons however they are expressed differently between organisms. So codons that are expressed more by E. coli should be chosen

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13
Q

What needs to be done to the plasmid supplied gene?

A

Amplification and cloning

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14
Q

What are the 4 steps in amplification and cloning?

A

1) Choose an expression plasmid
2) Amplify the lipase gene (GOI) using PCR
3) Clone the GOI into the expression plasmid to make a recombinant plasmid
4) Transform a strain of E. coli bacterium to express our GOI

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15
Q

What are the 4 things that an expression plasmid needs?

A

Multiple cloning site (mcs)
Antibiotic resistance marker
Induction elements
Purification tags

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16
Q

What is the mcs used for?

A

Multiple cloning site - RE (restriction enzymes) cleaves DNA into fragments at specific sequences called restriction sites. Forms overhangs in the DNA which allows recombination of GOI with high specificity

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17
Q

What is the antibiotic resistance marker used for?

A

Gives resistance to an antibiotic - only E. coli successfully transferred that contain the recombinant plasmid will survive

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18
Q

What are inducing elements used for?

A

Lac promoter is located upstream of our GOI - allolactose or IPTG will cause our GOI to be expressed

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19
Q

What are purification tags used for?

A

A His-tag (histodine) contains an imidazole side chain that can bind to nickel in a purification column

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20
Q

What is PCR?

A

Polymerase chain reaction - Used for amplification - creating large amounts of a specific section of DNA

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21
Q

What are the ‘ingredients’ required for PCR?

A
  • Genetic material
  • Short oligonucleotide primers
  • DNApol
  • Deoxynucleotide triphosphate monomers (dGTP, dATP, etc)
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22
Q

What does the GOI need to have to be incorporated into the expression plasmid?

A

Restriction sites that are complimentary to the expression plasmid - PCR primers must be designed to do this

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23
Q

What are the 3 steps involved in making forward primers?

A

1) Write out ~20 bases of the 5’ end of the lipase gene
2) Add the NdeI restriction site at the 5’ end (ATG) and a G/C rich region at the 3’ end
3) Add 8 bases upstream of the 5’ end

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24
Q

What are the 4 steps involved in making reverse primers?

A

1) Write out ~20 bp of the sense and antisense strand at the 3’ end of the lipase gene
2) Add the BamH1(GGATCC) restriction site at the 3’ end
3) Add 8 bases downstream at the 3’ end
4) Right out the antisense sequence in reverse with a G/C rich region at the 3’ end

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25
Q

What happens to double stranded DNA at high and low temperatures?

A

At high T - DNA melts and the strands separate
At low T - DNA strands reanneal

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26
Q

What are the 5 steps in PCR?

A

1) Primers, lipase DNA, dNTP’s and DNA polymerase are added together
2) Heated to high T (95c) - DNA melts
3) Cooled (68c) - Primers anneal to DNA
4) Heated (72c) - DNA polymerase extends primers
5) Heated to high T (95c) - DNA melts and the process begins again; exponentially amplifying the gene

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27
Q

What are the basic steps in cloning?

A

1) Digestion of the expression plasmid with restriction enzymes
2) Digestion of the PCR product by the same restriction enzymes to form overhangs that are complimentary
3) Ligation of the expression plasmid and the PCR product by DNA ligase to give a recombinant plasmid

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28
Q

What are the two types of cloning that we study?

A
  • Ligation Independent cloning (LIC)
  • In-fusion cloning
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29
Q

What are the advantages of LIC?

A
  • No custom restriction site designs
  • No DNA ligase requirement
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30
Q

How does LIC work?

A

1) Digest BseR1 site in LIC vector with BseR1
2) Treat with T4pol and dTTP - these digests the 3’ end until it meets a T base leaving a long overhang rich in G/C - LIC vector formed
3) Add G/C rich complimentary LIC sequences to the GOI
4) PCR - amplify
5) Treat with T4pol and dATP - single stranded overhangs complimentary to LIC vector
6) PCR product inserts iteself into the LIC vector without use of DNA ligase to from a recombinant plasmid

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31
Q

How does In-Fusion cloning work?

A

1) Digest (any) vector with a restriction enzyme to linearise it
2) Design PCR primers with 15 bp extensions that are complimentary to vector
3) PCR - with complimentary 15 bp to the vector
4) Recombinase enzyme recognises regions of homology between PCR product and vector and digests 3’ ends by 15 bases
5) Recombinase then binds the complimentary bases of PCR product and the linear vector

32
Q

What are the advantages of In-fusion cloning?

A
  • No separate treatment with DNApol and d(A or T)TP
  • No cleanup step after T4pol reaction
  • Can use any vector and any insert
33
Q

What is recombinant gene expression? (Heterologous gene expression)

A

Expression of a gene from genetically engineered DNA or expression of a gene from one organism in a different organism

34
Q

What operon is used in recombinant gene expression?

A

The lac operon

35
Q

Why is IPTG used for expression of the Lac operon?

A

It removes Lac repressor allow RNApol to bind a create T7pol and removes T7 repressor to allow T7pol to bind and express the GOI

36
Q

What does RNApol cause transcription of in recombinant gene expression?

A

T7 polymerase which expresses our GOI

37
Q

What is the procedure for IPTG-induced recombinant gene expression?

A

1) Transform E. coli with recombinant plasmid
2) Pick a colony of cells and grow cells in the present of antibiotic - only E. coli with recombinant plasmid will grow
3) Add IPTG to growth medium
4) Protein of interest is synthesized
5) Disrupt the cells (ultrasonication)
6) Centrifuge the extract

38
Q

What are the two layers after centrifugation?

A
  • Insoluble (membrane-associated)
  • Soluble (protein)
39
Q

What are the problems with heterologous gene expression?

A
  • No expression of gene (rare)
  • No soluble expression (protein is useless) ‘Inclusion Bodies’
40
Q

How can we improve soluble gene expression while using the same plasmid?

A
  • Temperature: at high temp proteins fold too quickly and form inclusion bodies
  • Chaperones: Proteins that help other proteins fold (co-expressed with the GOI)
  • Other strains of E. coli: some contain enzymes that help fold proteins at lower temperatures or enhance S-S bond formation
41
Q

How can we improve soluble gene expression by changing the gene?

A

1) Codon optimisation
2) Truncate the protein (remove signal sequences etc)
3) Use new tags (His-tag, MBP tag, GST tag)

42
Q

What is the final thing we can do to improve soluble gene expression?

A

Change the host organism (commonly Pichia pastoris - a yeast)

43
Q

Why is it difficult to express eukaryotic genes in prokaryotes?

A

Prokaryotes dont do post-translational modifications (PTM):
- Glycosylation
- Acetylation, formylation
- Disulfide bridge formations
- Phosphorylation

44
Q

What are the pros of using Pichia pastoris over E. coli?

A
  • Grows on methanol
  • High growth rate
  • Has two genes encoding the enzyme Alcohol Oxidase (AOX1/AOX2) (methanol acts as lactose)
  • Secretes proteins - disruption of cells is not needed
45
Q

What does a typical Pichia pastoris plasmid (pPicZ) contain?

A
  • AOX1 promoter
  • α-factor (signal sequence for secretion)
  • MCS (multiple cloning site)
  • His-tag (or other tag)
  • Antibiotic marker (zeocin)
46
Q

What is a complication when using Pichia pastoris?

A

It uses chromosomes not plasmids - amplification and cloning can be done in E. coli fortunately but an Expression plasmid cannot be used

47
Q

What are the three steps for cloning into Pichia?

A

1) make a cut in the MCS of pPicZ vector
2) insert the GOI using PCR
3) GOI is inserted next to AOX1 promoter, the alpha-factor and has a His-tag to form recombinant plasmid in E. coli

48
Q

What are the three steps for transforming Pichia?

A

1) Linierise recombinant plasmid
2) Transform Pichia cells with linearised plasmid using electroporation - a small electric shock that induces yeast cells to take up reombinant plasmid
3) Homologous recombination occurs - plasmid DNA inserted next to AOX gene in Pichia chromosome

49
Q

What are the two steps in expression in Pichia?

A

1) Inoculate on colony of yeast into YPD medium overnight
2) After 24h use this culture to inoculate larger culture in medium wiht 0.5% methanol as carbon source. Due to AOX this will cause expression of GOI not alcohol oxidase

50
Q

What are the two disadvantages of expressing in Pichia?

A

Proteolysis - proteases in Pichia will digest POI (can be fixed by using specially engineered strains of yeast)
Glycosylation - might be right for Pichia but wrong for POI

51
Q

What is SDM?

A

Site directed mutagenesis - selective changing of a gene sequence to change the amino acid sequence

52
Q

Why do SDM?

A

1) Allows investigation of the role of amino acids in a protein
2) Change the enzymatic properties of a protein

53
Q

What are the two things we need for an SDM experiment?

A

1) The encoding gene must be cloned and ligated into an expression plasmid
2) The structure of the protein must be known preferably with a ligand bound to identify the active site

54
Q

What are the 4 steps in an SDM experiment?

A

1) Primers must be designed for PCR - find the mutation site and design a primer that at its centre that has the altered codon of choice with 15bp either side
2) Run a PCR
3) After PCR extension there will be a mix of mutant plasmid and wild-type plasmid. Wild-type plasmid can be removed by DpnI which only digests methylated wild-type plasmids from E. coli
4) Transforming a strain of E.coli with the ‘nicked’ mutant plasmid will result in lots of repaired nicked plasmid being made. Miniprep the mutant plasmid and confirm mutation via sequencing

55
Q

What are the impacts of SDM?

A

It led to improving properties of industrially-significant enzymes like the serine protease: subtilisin

56
Q

What reaction does subtilisin catalyse? (What do serine proteases do?)

A

The hydrolysis of peptides (amide bonds) in the same way as lipases do with esters (nucleophilically activated serine residue). Prefers peptides that have hydrophobic groups (Tyr>Phe>Met>Ala) at the P1 position due to the Gly residue in the S1 sub pocket

57
Q

Where is the P1 residue site located?

A

To the left of the scissile bond (the one that is broken) which is the S1 sub pocket

58
Q

What are the adjectives of SDM experiments on SBL (subtilisin)?

A

1) increase stability to oxidants
2) change the substrate specificity
3) create the ability to create peptide bonds (instead of cleave them)

59
Q

Why and how is subtilisin engineered to have increased stability towards oxidants?

A

The sulfur containing sidechain Met222 (next to catalytic Ser221) is susceptible to oxidation by washing powders with bleaches causing denaturing and inactivation of subtilisin. Therefore it was mutated to Cys. This increases activity but is less stable compared to smaller residues for 0.1M hydrogen peroxide and much less stable for 1M hydrogen peroxide

60
Q

Why and how is subtilisin engineered to change its substrate specificity?

A

Mutations at subpockets changes the substrate specificity:
- Changing the Gly116 at S1 to more hydrophobic side chains increases activity with smaller hydrophobic side chains at P1
- Changing Ile107 at S4 to Gly reduces activity for small side chains at P4 as the cavity is now larger
- Changing residues at sub-pockets with basic amino acids bound to negative aspartate increased activity 60,000 times

61
Q

Why and how is subtilisin engineered to have the ability to create peptide bonds instead of cleaving them?

A

Introducing an N-terminus peptide to an esterified C-terminus peptide (instead of water) at the acyl enzyme intermediate causes the formation of peptide bonds. This can be favoured by mutating Ser221 to Cys221 resulting in a thioacyl enzyme intermediate as thiosesters are more sensitive to amines than water. Changing of Pro225 to Ala225 relieves steric crowding further increasing peptide bond formation

62
Q

What are the two things that we need to know about an enzyme before doing SDM?

A

1) Structure
2) Mechanism

63
Q

What is the natural process that occurs to improve or adapt enzyme functionality and what is the process called that replicates this?

A

Evolution - genetic mutation followed by natural selection
Directed evolution - generates random genetic diversity

64
Q

What are the two steps involved in directed evolution?

A

1) Generation of genetic diversity
2) Screening OR selection of isolated mutants of interest

65
Q

What are two ways of generating genetic diversity?

A

1) Error-prone PCR
2) DNA shuffling

66
Q

How does Error-prone PCR work (ep-PCR)?

A

Increasing [Mg+] and [dGTP] causes DNApol to make significantly more errors when transcribing RNA. After selecting the mutant with the desired properties the process can be repeated

67
Q

How does DNA shuffling work?

A

Mix closely related gene sequences with DNAase in a PCR machine to created single strands which melt, anneal and extend to form ‘mosaic genes’ with different properties

68
Q

What is the difference between screening, facilitated screening and selection?

A

Screening - all mutant genes survive and have the same phenotype so ALL MUST BE CHECKED
Facilitated screening - all mutant genes survive but sought characteristic genes have a different phenotype so CHECK ONLY PHENOTYPE
Selection - only mutant genes survive so CHECK SURVIVING

69
Q

What does an E-value represent for an enzyme?

A

The Enantioselective factor - how well an enzyme can convert one enantiomer over the other. 1 is poor, 50 is good, 200 is pretty good

70
Q

What are the three steps for the DE of PAL to improve its enantioselectivity?

A

1) Rounds of error prone PCR
2) Screening
3) Use of SDM to further improve E-value

71
Q

Where were the sites of mutagenesis in PAL and why where they surprising?

A

They were not in the catalytic centre but in other locations - shows that remote effects improved E-value. We would not have changed these residues using SDM - ED allows us to irrationally come to a useful product

72
Q

What are the two mechanisms that haloalkane dehalogenase catalyzes and how did ED change its efficiency?

A

1) Ring closing of halohydrin to form epoxide
2) Ring opening of epoxide with cyanide to form cyanohydrin
Improved these reactions 4400-fold by causing 35 mutations

73
Q

Why can ED be more useful in SDM when trying to improve a characteristic of an enzyme?

A

SDM requires understanding of both the structure and mechanism of an enzyme to alter a specific residue rationally, whereas ED can leaved to irrational mutants that can increase the required characteristic

74
Q

What is genome editing and what is the superior system that does this?

A

The addition and removal of genes - CRISPR-Cas9 system

75
Q

What does CRISPR do?

A

It inactivates unwanted incorporated foreign DNA from viruses or phages

76
Q

How does CRISPR work in nature?

A

1) Invasion DNA is incorporated into genome with short sections of repeating DNA for protection against future invasion
2) Phage DNA invades again
3) Bacteria transcribes old viral DNA segment resulting in the Cas protein with RNA that binds to Cas (tracrRNA) and crRNA with a complimentary sequence to the invading DNA
4) crRNA binds to invader and Cas cleaves it, inactivating it

77
Q

How is CRISPR-Cas9 exploited?

A

Single-guide RNA is designed so that it is complimentary to the region of gene that is to be removed. Introduced via plasmid with Cas9 nuclease which when transcribed/translated results in sgRNA/Cas9 complex which binds to DNA and causes inactivation of targeted gene

Genes to Proteins (4 decks)

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