Genetic Engineering Flashcards
How do you determine mathematically the possibilities of an amino acid sequence?
n^20
where n is the number of amino acids in the sequence
What is Transcription?
The process whereby DNA is converted into mRNA
What is Translation?
The process whereby mRNA is translated into the amino acid sequence on the ribosome, giving a protein product
Describe the mechanism of lipase catalysed ester hydrolysis
1) histidine deprotonates the serine (active site), which attacks the electrophillic carbon of the ester
2) A tetrahedral oxyanion is formed; this stabilised by NH groups from the backbone of the protein
3) The departing alcohol picks up a proton from the histidine and an acyl enzyme intermediate is formed
4) Water is now bound in the active site, this is deprotonated (activated) by the His224 residue and attacks the C=O of the acyl enzyme intermediate
5) another oxyanion formed, which collapses with the loss of the carboxylic acid product, to regenerate the active site
Define kinetic resolution in terms of racemic esters interacting with lipase
When presented with a racemic ester, lipases only hydrolyse one enaniomer
What are the ways of obtaining a specific gene?
1) Obtain the ‘genomic DNA’ of the organism
2) Have the gene synthesised - most common
What things are to be considered when synthesising a gene?
1) find the gene sequence
2) in eukaryotic cells - check for introns, if available use. the mRNA sequence for gene design
3) check for flanking (useless) N-terminal additions as signal sequences
4) for E. coli - Codon optimise
How do you go about cloning your gene once you have it? (4 steps)
1) Decide on an expression plasmid
2) Then amplify the lipase gene (GOI - Gene Of Interest) using PCR (Polymerase Chain Reaction)
3) Thirdly clone the gene into the expression plasmid, enabling selective manipulation + expression - Gives a recombinant plasmid
4) Recombinant Plasmid containing lipase gene then used to transform a strand of the E. coli - used to produce our lipase from the gene
What is an expression plasmid?
The vector used to carry the gene that encodes the lipase into the E. coli bacterium to make the proteinn
What are the features of an expression plasmid that help enable coding and gene expression?
Multiple Cloning Site (MCS)
Restriction Enzymes (RE)
Antibiotic Resistance Marker
What are Multiple Cloning Site (MCS) and Restriction Enzymes (RE) ?
MCS has restriction endonuclease sites that permit cloning
RE cleaves DNA into fragments within dsDNA called restriction sites - ‘sticky ends’ or ‘ overhangs’
What is an antibiotic resistance marker?
Allows selection of only colonies of E. coli that contain desired plasmid - only these survive
What is the system in an expression plasmid to induce expression?
lac promoter is upstream of the gene we want to express, but NOT the beta-galactosidase gene
- addition of lactose (IPTG) to growing cells will induce expression of out GOI (Gene Of Interest)
How can we facilitate the purification of our protein?
Having the ability to introduce TAGs - like a polyhistidine tag
Near the 5’ end, 6 codons can be put for the amino acid residue Histidine, which has an imidazole side-chain which is very good at chelating metals,
therefore after our protein is produced, one step purification by column chromatography with beads loaded with Ni2+ ions, only His-tagged protein will bind
then increase concentration of free imidazole to out compete the protein for the beadsd
What are the 4 ingredients for the Polymerase Chain Reaction (PCR)?
Genetic Material - containing the gene to be amplified
Short olgionucleotide primers - complementary to the 5’ and 3’ sequences of the gene to be amplified
DNA Polymerase - catalyse the extension of the growing DNA chain (also some Mg2+ ions for funsies)
Deoxynucleotide Triphosphate monomers - dGTP, dATP, dCTP, dTTP
What are the 5 steps of PCR?
1) Ingredients: olgionucleotide primers, template DNA, dNTPs and polymerase with Mg2+
2) Heated to 95C, dsDNA melts
3) Coolled to 68C, short olgionucleotide primers anneal to the separated strands
4) Heated to 72C - optimum temp for DNApol - extends the DNA chain on each strand using dNTPs
5) Heated to 95C again, the new double strands are melted, new primers anneal and extend… number of new copies doubles every cycle…. exponential amplification of the gene
Where is PCR performed?
A thermal cycler
What are the 3 steps of ‘tradtional’ cloning?
1) Digest the expression plasmid with restriction enzymes, with designed sites in the PCR primers
2) Digest the PCR product, with the same restriction enzymes - leaves complementary overhangs
3) Ligate the ‘insert’ and the plasmid together, using DNA ligase and the cofactor ATP - gives a recombinant plasmid
What are the advantages of Ligation Independent Cloning (LIC)?
1) No custom restriction site design requirements
2) No DNA ligase requirement
Describe the steps of Ligation Independent Cloning (LIC)
1) Digest vector - give short GG and CC overhangs
2) Add T4DNApol in dTTP - Eats at GC until T - then stops digestion
3) Mix vector with long single stranded overhangs - ligate spontaneously
Why is ligation spontaneous in Ligation Independent Cloning (LIC)?
H-bonding affinity between C and G bases is so strong
(is very G C rich)
What are the advantage of In-Fusion Cloning?
Can be used with any insert and any vector
No separate treatment of precursor fragments with DNApol/dATP
No clean up step after T4pol reaction
What are the 5 steps of In-Fusion Cloning?
1) Digest vector with restriction enzyme - linearises it
2) Design PCR primers with 15 base-pair extensions complementary to vector
3) Recombinase recognises broken dsDNA and acts as a 3’-exonuclease ‘eats’
4) single stranded overhangs are stabilised by ‘single-stranded’ binding protein
5) DNApol holds the single strands in position for H-bonding - delivers intact recombinant plasmid
What are the 5 steps for IPTG-induced heterologous expression?
1) Transform E. coli with recombinant plasmid and incubate on agar overnight
2) Pick a colony of cells and grow in presence of antibiotic
3) add IPTG
4) IPTG binds to lac repressor protein - released from lac promoter, gene expression initiated by mRNA synthesis by the binding of RNA polymerase
5) Gel analysis shows protein is being produced
What may happen if the cells are disrupted in gene expression with centrifugation?
you get:
INSOLUBLE fraction
and a
SOLUBLE fraction (want protein to be here)
What if there is no expression or no soluble expression in gene expression?
As the protein emerges from the ribosome, it is bound up and unfolded (USELESS)
-aggregates as Inclusion Bodies
How can we improve soluble gene expression?
Using the same plasmid-
- temperature control - slow the rate of protein folding off the ribosome
- chaperones - proteins that aid in folding
- different strains of E. coli
If nothing else works, what else can we do to re-engineer your gene?
1) codon optimisation
2) make truncated protein
3) new tags for solubilisation
- use protein that binds to column media, then elute and cut off tag with enzyme
What are alternative Host Organisms for Gene Expression?
1) Other Bacteria
2) Yeasts
3) Fungi
4) Insect Cells
What are Post-Translational Modifications?
Altering proteins after translation:
-Phosphorylation
-Acetylation, formylation
-Formation of disulphide bridges
What does a typical vector for cloning into P. pastoris (pPICZ) contain?
- AOX1 promoter
- Multiple Cloning Site (MCS)
- A sequence containing purification tag (his-tag)
- Antibiotic Marker for P. pastoris - ZEOCIN
What are the 3 steps for cloning into Pichia Pastoris?
1) Make a cut in the MCS
2) Insert Gene of Interest (GOI) - using regular cloning (LIC, InFusion)
3) Gene is inserted adjacent to AOX1, alpha-factor has a hexahistidine tag
What are the 3 steps for transforming Pichia Pastoris?
1) Linearise recombinant plasmid
2) Transform Pichia Pastoris cells with the linearised plasmid using ‘electroporation’
3) Homologous Recombination occurs, plasmid DNA gets inserted into the AOX gene
What are the 2 steps for expressing in Pichia Pastoris?
1) Inoculate one colony of the yeast into YPD medium overnight
2) After 24 hr growth, this small culture is used to inoculate a larger culture in medium with 0.5% methanol (carbon source)
What are the disadvantages of expressing in Pichia Pastoris?
Proteolysis - Pichia has many proteases that will digest your protein,, but these can be knocked out by specially engineered strains
Glycosylation - May gave to de-glycosylate your protein using enzyme
What is Site-Directed Mutagenisis?
A technique that allows us to selectively change the gene sequence within a gene - allows us to change the amino acid sequence
What are the benefits of Site-Directed Mutagenesis (MDS)?
1) We can investigate the role of amino acids in the mechanism of a protein
2) Can change enzymatic properties for biotechnology
What 2 things are needed for Site-Directed Mutagenesis (MDS)?
1) The gene encoding the enzyme must be cloned and also ligated into an expression plasmid, allowing easy recovery of pure protein
2) Know the structure of the enzyme - preferably with a ligand bound - in order to identify the active site
What are the 5 steps of Site-Directed Mutagenesis (MDS)?
1) Mutation via PCR
2) First design primers for PCR - find required mutation site; design primer with a centre of the chosen anticodon - 15bp either side
3) Run PCR
4) Remove wild-type template plasmid with enzyme DpnI
5) Transform a strain of E. coli with ‘nicked’ mutant plasmid from PCR - results in bacterium making lots of copies - Then miniprep the mutant plasmid - confirm mutation via sequencing
What is Subtilisin?
A serine protease
What are the features of subtilisin?
-Single Polypeptide Chain
-No dishulphide bridges - stable and easy to express
-Catalytic site has a triad of amino acid residues (Asp32, His64 and Ser221)
-Shallow binding groove for subtrates
What reaction does subtilisin catalyse?
The hydroylsis of peptides -
Tyr>Phe>Met>Ala
How can Subtilisin be engineered to provide greater stability towards oxidants and why is it needed?
Enzymes are susceptible to denaturation by oxidation.
In the active site, Met222 contains sulphur which can be oxidised.
Met222 can be mutated to Cys - increases activity but is very susceptible to hydrogen peroxide - need a hydrophobic side chain instead
How can Subtilisin be engineered for substrate specificity and why is it needed?
In wild-type Subtilisin, the bottom of specificity subpocket S1 contains Gly166 so that large hydrophobic side chains are accommodated.
If you mutate at position 166, you can introduce larger, more hydrophobic side chains
How can subtilisin be engineered to synthesise peptide bonds?
We need to introduce a peptide with an amino terminus to attack the Acyl Enzyme Intermediate (AEI) instead of water and form the bond (ligation)
How can we favour ligation over hydrolysis?
Mutate Ser221 to Cys
Further improvement can be mutating Pro225 to Ala which relieves steric crowding in the active site
Describe directed evolution for engineering by Haloalkane Dehalogenase (HHDS)
HHDH is able to catalyse:
1) the ring closing of the halohydrin to form the epoxide
2) the ring opening of the epoxide with cyanide to form the cyanohydrin
What is CRISPR-Cas9?
CRISPR-Cas9 system - a DNA repair system in bacteria that is used to deactivate by hydrolysis unwanted foreign DNA from infection
Describe the conversion of Glucose to DHAP
Glucose is phosphorylated, isomerised, phosphorylated again into fructose-1,6-diphosphate
- Cleaved by aldolase enzyme into to C3 components - one is DHAP
Describe the conversion of DHAP to Glycerol
In the engineered strain off E. coli:
2 genes encoding enzymes are engineered into the E. coli
- G3P dehydrogenase, converts DHAP to glycerol-3-phosphate
- G3P phophatase, converts glycerol-3-phosphate to glycerol
describe the conversion of glycerol to 13PD
In the engineered. E. coli:
Two genes encoding enzymes are engineered into the E. coli
- Glycerol Dehydratase, converts glycerol into 3-hydroxypropionaldehyde
- 1,3-Propanediol Oxidoreductase, converts 3-hydroxypropionaldehyde into 13PD
