Molecular Genetics 25-30 Flashcards
Why do we use model organisms?
- Long history of use
- Easy to grow and maintain in the laboratory
- Easy to manipulate
- Short life cycle
- Small genome
- Sequenced genomes: ideas of what caused which phenotypes
- Many mutants
- Used to inform the biology of other species
- Extrapolate from simple organisms to more complex ones
- Exploited in biotechnology
Features of E. coli
- Escherichia coli
- Main organism used to manipulate DNA
- Rod-shaped
- Lives in colon - pathogenic
- Rapid growth
- Stored in fridge
- Model PROKARYOTE
What are examples of model prokaryotes?
E. coli
Bacillus subtilis
Who transformed the first recombinant DNA into a prokaryote and when?
1972
Boyer and Cohen made the first recombinant DNA and transformed it into E. coli
When was E. coli’s genome sequenced and how many genes does it have?
Sequenced in 1997
4000 genes
Features of bacillus subtilis
- Rod-shaped
- Gram positive
- Found in soil and digestive system of ruminants
- Forms tough spores
- Commensal
- Used to study bacterial cell differentiation and chromosome replication
- Secretes proteins easily
- Used in biotechnology in large scale enzyme production
What are examples of model unicellular eukaryotes (yeasts)?
Saccharomyces cerevisiae
Pichia pastoris
Features of Saccharomyces cerevisiae
- First eukaryote sequenced (1996)
- 6000 genes
- Yeast doubles every 90 minutes
- Stored in fridge
- Has several plasmids
What are 3 plasmids that Saccharomyces cerevisiae possesses?
- 2u plasmid (high copy) (YEp): plasmid used for cloning in yeast
- YAC (yeast artificial chromosome) plasmid (low copy) (YCp/CEN):
- Integrating plasmids (YIp)
Features of pichia pastoris
- Used in protein production
- Similar growth conditions as S. cerevisiae
- Grows to a high density
- Sequenced genome (2009)
- Often used to produce large quantities of proteins for purification for analysis
- Vectors integrate into genome
What does YEp stand for as the other name for yeast’s 2u plasmid?
Yeast episomal plasmid
General properties of modified plasmids (basic cloning vector)
- Small (3-10kb)
- Gene for antibiotic resistance
- Easy to transfer from cell to cell
- Easy to isolate from host
- High copy number (100-150 copies of plasmid per cell)
- Easy to detect and select
- Multiple cloning site / polylinker where restriction enzymes can cut and inset DNA
- Able to screen recombinants
Types of cloning vector
- Modified yeast plasmids
- Artificial chromosomes
- Lambda replacement vectors
- Cosmids and Bacmids
- Ti plasmid
Properties of BACs, YACs and PACs (artificial chromosomes)
- Bacterial artificial chromosomes
- Yeast artificial chromosomes
- P1 bacteriophage artificial chromosomes
- Can insert really large DNA fragments (up to 2000 kb)
Which out of PACs, BACs and YACs can take the largest fragment?
YACs
What is an important feature of YACs (CENs)
They are shuttle vectors, meaning they can be shuttled between two types of organisms
Example: using YAC as a shuttle vector between yeast and E.coli
- Two selectable markers (for yeast - auxotropic marker and E. coli - antibiotic resistance) required
- Need two open reading frames
- Plasmids will be circular in E. coli and linear in yeast
Features of Yeast Integrating Plasmids (YIp)
- In S. cerevisiae
- Useful for stable integration of something into the yeast DNA
- Do not replicate independently
- Recombines into chromosome - to select the chromosome will have the non-functional version of a gene and the plasmid will have the intact version, which is how you select for if the chromosome has taken up the plasmid
- Transformation frequency low, but transformant is stable
Features of lambda replacement vectors (aka bacteriophage vectors)
- Bacteriophage is pathogen of E. coli
- Has linear dsDNA with two cohesive ends (cod sequences) which attaches to E. coli cell and injects DNA in
- DNA is linear when packaged into protein head, with cohesive ends on each end. Once in the cell, these cos sequences are removed and DNA circularises, replicated and produces more phages which cause the cell membrane of E. coli to lyse
- Middle section removed and replaced with gene of interest
- Larger than typical cloning vector (37-52 kb)
- Difficult to manipulate
- Used in cDNA and genomic libraries
Features of cosmid vectors
- Just highly modified lambda replacement vector
- Everything between cos sites is removed
- DNA up to 45kb can be inserted
- This is packaged up into lambda particles and then used to infect E. coli
What is gateway cloning?
- Developed in the 1990s
- Uses lambda replacement vector to transfer between vectors
- Lambda can integrate into the genome
- Integrase cuts at two sites: attP (in the plasmid/phage DNA) and attB (in the bacterial DNA)
- DNA will then ligate and is inserted into the chromosome - the sites are then called attL and attR
- This reaction is REVERSIBLE due to Xis excisionase. This is what gateway cloning exploits
Process of gateway cloning
- There are two vectors involved: an entry vector and a destination vector
- The entry vector is a basic cloning vector with a selectable marker (eg kanamycin), an origin of replication and a MCS. Gene of interest put into entry vector with BP clonase
- Instead of lacZ gene in the vectors, a toxic ccdB gene is used. E. coli will be killed if there is not an entry into the ccdB gene as the E. coli will express the toxic gene
- Gene of interest switched into destination vector using LR climate
When is it better to use a shuttle vector for cloning rather than gateway cloning?
When you need to clone more than 4 fragments, as the gateway cloning method only allows you to clone up to 4 DNA fragments
Process of cloning more than 4 fragments
Using a shuttle vector (usually 2u plasmid) which shuttles from yeast to E. coli
Vector has two selective markers and two origins of replication (one for E. coli one for yeast)
If you add 2u and yeast selectable markers you can get any plasmid to function in yeast
Outline vector construction in yeast
Use a 2u plasmid eg pRS426
1. Linearise fragment
2. Amplify fragments with complementary overhangs by PCR
3. Transform into yeast and yeast will assemble fragments
Can take 3 days - quite quick
Outline the transformation of yeast
- Use LiAOc (lithium acetate) to make yeast cells competent
- Add PEG (polyethylene glycol) and plasmid and PCR products
- Allow to recover at 30 degrees
- Heat shock at 42 degrees
- Plate out for 3 days on selective medium
- Recover plasmids from yeast via miniprep (need and enzyme to digest cell wall so plasmids can be removed)
What does competent mean?
When a cell becomes able to alter its genetics by taking up extracellular DNA from its environment and become transformed
Outline Gibson assembly
- Design primers to add overhangs
- Add master mix provided by kit and incubate
- An exonuclease will remove the 5’ ends so fragments can anneal
- Close and seal gaps with DNA polymerase and DNA ligase
When was yeast first transformed?
1978
How many fragments can the Gibson assembly clone?
Up to 15
Outline Golden Gate assembly
- Used type IIS restriction enzymes
- These cleave outside of their recognition sequence, creating four base flanking overhangs
- Add type IIS sites to primers and amplify your product
- The destination vector contains sites with complementary overhangs
How many fragments can golden gate assembly clone?
8-10 fragments
What type IIS restriction enzyme is often used in golden gate cloning?
Fok1
Recognised the asymmetric sequence GGATG and makes a staggered cut further down the sequence
Outline the transformation of E. coli with the cloned vector
- Make the E. coli competent
- Incubate the E. coli to give it time to produce the proteins for antibiotic resistance or another marker
- Plate the E. coli on a plate containing antibiotics
What are the two ways to make E. coli competent?
- Permeabilise the cell wall using calcium chloride, keep on ice, heat shock
- Electroshock cells - this also permeabilises the cell wall
How to extract and store the plasmid?
- Screen E. coli colonies (eg blue/white screening, colony PCR where you look for the presence of certain fragments)
- Do an E. coli miniprep to extract plasmid
- Verify plasmid by PCR, sequencing and digestion
- Store plasmid DNA at -20 degrees
- Store miniprep E. coli culture in glycerol and freeze in -80 degrees
- Repeat this process to produce more plasmid
What is the alternative to a miniprep if you need more plasmids than it can supply?
Do a midiprep
What are 3 model fungi?
Aspergillis niger, A. oryzae and A. nidulans
Features of model fungi
- Human pathogens (Aspergillosis = respiratory disease), mildew, used in fermentation
- Genomes sequenced in 2007, 37 mb
- Grow in 3-5 days
- Transformation is via electroporation (electric shock to take up DNA), PEG-mediated or Agrobacterium
How is citric acid produced today?
With Aspergillus niger
What was the old method of producing citric acid?
Calcium citrate produced and then converted to citric acid chemically
What microbe produces monosodium glutamate (MSG)?
Corynebacterium glutamicum
What is Corynebacterium glutamicum also used to produce besides MSG
Lysine - an essential amino acid only found in small quantities in cereal crops. It is added to animal feed. Mutants were found that accumulated lysine under limiting conditions, but the enzyme that synthesisers lysine is inhibited under high lysine conditions. New mutants were found where this inhibition was defective.
Also used in production of methionine
Why is the chemical synthesis of vitamin C not preferred?
Chemical synthesis produces D- and L- forms of amino acids
Humans can only taste L-forms and only L-forms can be incorporated into proteins, so half of the product is wasted
Why is the biological synthesis of vitamin C preferred?
Microorganisms only produce L- forms
What does the bacterium Gluconobacter oxydans do?
Convert sorbitol to sorbose - an intermediate step in the process of producing vitamin C
What was the process devised in the 1930s of producing vitamin C
Combination of chemical and biological methods to produce vitamin C
- Nickel catalyst produces D-sorbitol
- G. oxydans converts sorbitol to sorbose
- Acid converts sorbose to 2-KLG
- 2-KLG concerted with an acid to vitamin C
What is the starting mixture used to produce vitamin C?
Glucose
Outline the modern process of producing vitamin C
- The bacterium Erwinia sp. can convert glucose to 2.5 DKG
- Corynebacterium sp. can convert 2.5 DKG to 2-KLG
- Problem: different growth conditions needed
- Solution: transfer the gene from Corynebacterium sp. into Erwinia sp. so only one growth condition needed
- Acid treatment then converts 2-KLG to vitamin C
Advantages of recombinant protein production using E. coli
- Simple to grow
- Can grow in large bioreactors
- Cheap
- No ethical issues
- Safe
- High yields
Disadvantages of recombinant protein production in E. coli
- Codon bias
- High production can be toxic: kills E. coli culture
- Poor folding which leads to inclusion bodies being produced
- Poor secretion
- Post-translation differences
Why is codon bias a disadvantage of using E. coli to produce recombinant proteins? What is the solution?
- Redundancy in genetic code
- In E. coli, AAA is used to code for lysine most of the time
- If the sequence contains AAG instead, low amounts of the protein will be produced
- This can be overcome by site-directed mutagenesis of the DNA sequence, or synthesising the DNA, or feeding the culture with a supply of the missing tRNAs, or transforming the relevant genes into strains of E. coli
- Codon optimisation improves yield by about 70% when expressing human proteins in E. coli
Why is poor secretion a disadvantage of using E. coli to produce recombinant proteins? What is the solution?
- E. coli secretes few proteins
- Secretion into the culture medium facilitates protein recovery
- This is done by fusing the protein to a protein that is usually secreted or adding additional secretion sequences
- You could also modify E. coli to have a better extracellular secretory system
Why are post-translation modification issues a problem when using E. coli to produce recombinant proteins? What is the solution?
- Glycosylation is required for the proper function of many proteins
- Adding sugar residues to protein
- Prokaryotes add glycosyl groups to side chains, rather than N-terminal groups which is where eukaryotes add them
- The prokaryotes are therefore adding the glycosyl groups to the wrong place for human proteins
- The solution is taking glycosylation genes from Campylobacter and adding them to E. coli so your proteins will be glycosylated correctly
Insulin production in E. coli (old method)
- Clone and express genes for insulin strands A, B and C in E. coli
- This left preproinsulin with no post-translational modification
- Put gene for A and B chains on two separate plasmids and put these into separate E. coli. Fused the proteins to B-galactosidase to ensure chains were secreted from the cell
- Once proteins were collected they were chemically treated with cyanogen bromide to cleave off B-galactosidase
- Oxidisation step required to form disulphide bonds and produce active insulin
Natural insulin production
- Insulin strands B, C and A translated to give linear peptide
- C strand cleaned off
- Disulphide bonds form between strand A and B, leaving the complete insulin molecule
Advantages of using yeast to produce insulin
- Simple to grow
- Can grow in bioreactors
- Cheap
- No ethical issues
- Safe
- Glycosylates secreted proteins
- Improved folding - disulphide bonds form
Modification of porcine insulin for human use
- Allergenic reactions to porcine/bovine insulin
- Use enzymes to modify protein
- Trypsin cleaves next to arginine and lysine
- In porcine insulin, the terminal alanine is preceded by lysine
- Trypsin replaces the cleaved alanine with threonine - makes it into human form of insulin
Modern way to product insulin in E. coli
B, C and A strands synthesised, cloned in one plasmid and produced in one culture to produce insulin
Proinsulin digested with tryposin to remove C peptide and yield insulin
Disadvantages of using yeast to produce insulin
- Low yield
- Poor secretion
- Introns not spliced out as yeast is prokaryote
- Excessive glycosylation
What is yeast used to produce (medically)?
- Insulin
- Growth factors
- Blood clotting factors
- Viral proteins used in vaccines
How to solve the problem of insulin being prone to clotting?
- Clumping covered the receptor bonding site so reduces efficiency
- Not a problem in vivo as is secreted before it can clump
- Problem when injected
- Replace proline with asp
- These amino acids have negative charges so repel each other, preventing clumping
Features of Arabidopsis thaliana
- Thale cress
- Most widely used model in plant genetics
- Similar genetic responses to stress and disease as food crops
- Genome sequenced in 2000, 25,000 genes
- 5 chromosomes
What makes A. thaliana such a useful model plant?
6-8-week life cycle
Easily transformed
Many seeds produced
What are the two species of the second model plant genus?
Nicotiana benthamiana
Nicotiana tabacum
What features make Nicotiana sp. such useful model organisms?
- Often used in virology and tissue culture
- Easy to cross
- Thousands is seeds per cross
- 3-4 month life cycle
How to make a transgenic plant:
- As plant cells are totipotent, you can regenerate a whole plant from one cell
- These cells are grown in tissue culture
- There are various methods of transformation
What are the 3 methods of tissue culture to clonally propagate plant cells?
- Plant leaf discs: take section of leaf and growing it in a petri dish. Hormones required to stimulate growth of shoots and roots
- Callus capture: a mass of undifferentiated cells from an immature embryo or meristem, also treated with hormones to induce growth
- Suspension culture: important for producing recombinant proteins. Protoplasts (call wall removed) or immature pollen placed in suspension and grown for several years in culture
Features of Agrobacterium tumefaciens
Plant pathogen
Gram negative bacteria
Soil dwelling
Causes crown gall
Attracted by phenolics and enters through plant wounds
Induced plant to produce excess auxin and cytokinin
Galls contain opines synthesised by genes on the Ti plasmid