Albert Bolhius: Recombinant Dna Technology Flashcards
What are e coli and recombinant plasmids mixed with to allow plasmids to be taken up?
Mixed in the presence of CaCl2 and heat pulse
Somehow this creates pores and allows plasmids into the E coli
What bacteria is initial cloning stages usually done in?
E.coli
Recombinant plasmids mixed with it, and E coli them cultured
In PCR, what temp is desaturation of DNA strands (separation) done at?
94 degrees, takes around 30 seconds
What temperature is annealing with primers done at?
Cooled to 55-65 degrees from 94 degrees that was needed for desaturation, primers need this cooler temp to anneal, only takes 30 seconds
What temp is elongation with DNA polymerase done at?
72 degrees
DNA polymerase is thermostable
Takes 1 min per kilo base made (per 1000 bases)
When forming a DNA library, what vector is sometimes used? Why?
Phage ʎ
These can accommodate larger fragments than plasmids
But plasmids may still be used
What are the four ways to screen genomic libraries? Tricky to remember!!
Protein activity screening. ONLY works if activity of protein is easily measured.
Functional complementation: just remember you’re using strands of DNA you know are complementary to the gene you’re looking for that’s somewhere in the DNA library!
Hybridisation using DNA labelled probes, need info on gene sequence of interest to make the probe.
Immunological screening, using antibodies that bind proteins of interest.
What’s DNA sequencing used for?
Used to determine the order of nucleotides in DNA, ie determine the SEQUENCE
Used for:
Identifying sequence for drugs to target
Identifying mutated sequence that cause disease
Checking cloning experiments have worked propley
Understanding how genes function
How is DNA sequencing carried out?
A, T , C and G REACTIONS occur, whereby your sequence is cut/ fragmented at each one of these bases!
This forms every fragment possible
These fragments ran through sequencing gel (DNA electrophoresis)
Smaller molecules move fastest, to end of plate, larger molecules move slower.
Read sequence from bottom of plate!
The A,T,C and G reactions in DNA sequencing, where the sequence is fragmented at each base, can be done chemically or enzymatically.
What is the name of the chemical method?
What is the name of the enzymatic method?
Chemical: Maxam and Gilbert
Cleavage of bases here uses chemical reactions
Enzymatic: Sanger sequencing
How does Sanger sequencing work? What’s the enzyme used?
The enzyme DNA polymerase incorporates ddNTPs (deoxynucleotides) into the sequence (chain)
Whenever one of these ddNTPS (eg ddATP, ddGTP, ddCTP) is incorporated into the chain, the reaction stops and a FRAGMENT forms, as the chain is terminated
Eventually get a fragment at every single base
Run these through gel electrophoresis, seperates fragments and you can read the sequence from the bottom.
With the Sanger sequencing method, how can the nucleotides ddATP, ddGTP, ddCTP ddTTP be labelled? (hint: 2 options)
Radioactive labelling using radioactive isotopes attaching to diff nucleotides.
Fluorescent labelling, each of the four labelled with four diff colours . A computer reads the sequence.
What is bioinformatics?
You are translating a DNA sequence into its amino acid sequence.
You then compare the sequence translated with a data base (BLAST-N, BLAST-P, BLAST-X)
Then you can compare sequences to for eg find mutated genes, or identify similar sequences in diff organisms.
In bioinformatics, when translating the sequence, how many different reading frames are possible?
3 per strands; 2 strands so 6 reading frames in total.
Only one of these reading frames usually contains correct gene.
Start reading cram with start codon ATG
When viral DNA gets into one of our cells, enzymes will digest this DNA. Why don’t these digest enzymes digest out own DNA?
Some enzymes modify our own DNA by adding a methyl group.
When the methyl group is present the restriction enzymes won’t be able to cut the DNA.
Where do restriction enzymes cleave? What are these sequences called?
Cleave at palindromic sequences (reads the same forward and backward) this can create blunt or sticky ends
Does the EcoRI leave blunt or sticky ends?
Sticky ends
Enzyme from e coli
Restriction enzymes cut,
What enzymes paste?
Dna Ligase
It’s an ATP dependent enzyme
Which has better resolution, polyacrylamide gel or agarose gel?
Polyacrylamide
Agarose gel easier to use, allows you to run through bigger fragments
What is the typical DNA digest mixture composed of that you’d run through gel electrophoresis for 60 min at 60-129 volts?
DNA Water Concentrated buffer Restriction enzyme (also need a molecular weight marker, and after gel has been run, need to stain with a fluorescent marker and visualise under UV light)
When a DNA digest using restriction enzymes has been ran on a gel electrophoresis, and fragments have seperated, how do you determine size on fragments?
Bands visualised under UV light
Marker bands, of a known size, are held against the bands on your gel fro reference. You can read off sizes.
For a ligation reaction, what does the mixture require?
Vector
Insert (fragment produced by your restriction enzyme)
Buffer
Water
Ligase enzyme
Restriction enzyme to cut vector (SAME restriction enzyme used to cut fragment!)
When using dna ligase, The ends of the DNA fragment and ends of the cut vector must be complimentary, how’s this achieved?
Used the SAME restriction enzyme to cut the vector and make the DNA fragment inserts.
It will locate the same restriction site and therefore cut producing complimentary ends
What kind Of bonds does the Ligase form between the insert and the vector?
Covalently linked
by 3’-5’ phosphodiester bonds of the DNA
Can two different fragments digested by two different restriction enzymes possibly Ligate together?
Yes they can
Providing the sticky ends are the same sequence
Two different restriction enzymes may cut the same sticky ends
What is Phage ʎ?
A vector
It’s a virus that infects bacteria, therefore can infect e.coli with recombinant DNA
This is more efficient than plasmids infecting e coli
Accommodate large inserts
Good for DNA libraries
What 3 things do plasmids have that make them good for cloning?
Replication origin (allows new copies of plasmid to be made) Selection marker (eg genes for antibiotic resistance, important for cloning and seeing which plasmids have taken up what) Regions where DNA can be inserted
What does the plasmid pUC18/19 contain?
Multiple cloning sites
Genes for ampicillin selection
Ori for replication
Reporter gene Lac Z
When carrying out DNA recombinant technology, cells are usually mixed with ampicillin at some point. What is this for?
So that the selection of cells that took up the plasmid can be identified, as all plasmids will contain the gene for ampicillin resistance, therefore cells that die on the ampicillin plate didn’t take up a plasmid.
What are shuttle vectors?
Plasmids that are able to replicate in at least two different hosts
What are integration vectors?
Plasmids that can’t replicate in a particular host, but they will integrate themselves into the chromosome
What are artificial chromosomes? Are they plasmids?
Based on bacterial (BAC) or yeast (YAC) DNA.
They’re NOT plasmids
Known as BACs and YACS
Can contain very LARGE inserts (like phage)
Plasmids can only accommodate small inserts
Does bacterial DNA contain introns?
No, therefore it can be cloned directly,
Eukaryotes DNA does contain introns however, problems when it comes to cloning as bacteria can’t splice out these introns
Remember: eukaryotic genes with introns are NOT functional in bacteria
What happens to the poly A tail of mRNA each time its transcribed?
The polyA tail is shortened
When it becomes very short, the mRNA strand will degrade
This is why we say mRNA is very unstable
Eukaryotic genes with introns are NOT functional in bacteria, so what is done?
DNA copies of mRNA Are made for cloning in bacteria. This is called cDNA (complementary DNA). This doesn’t contain introns.
So this is still DNA, but without introns, formed from copying mRNA
How is mRNA isolated from all other RNA in the cell, needed to make cDNA?
All the cytoplasmic RNAs are combined with oligo(DT) matrix, under hybridisation conditions.
This matrix will only bind to the poly A tails of mRNA.
tRNA and rRNA can then be washed away, and mRNA is left over and purified
What enzyme is needed to convert RNA into DNA strands?
Reverse transcriptase
dNTPs also need to be added so the enzyme can use these to make the new DNA strand
How do you synthesise the second strand of DNA once cDNA has been formed from RNA?
Firstly remove RNA strands using an alkali
Add a poly(dG) tail, use terminal transferase and dGTPs, add this tail to the cDNA strand that’s already been formed.
Then hybridise this with an oligo-dC primer; this primer will attach itself to the poly G tail. Thus creates a small double stranded section of DNA which means a second strand can be synthesised!!
This creates double stranded cDNA
What do you do with cDNA once it’s been formed?
Can then be cloned in a vector such as a plasmid and put in bacteria, as this DNA will have had its introns removed!
What’s terminal transferase needed for when synthesising ds cDNA?!
Needed to add poly G tail to cDNA strand so that the oligo- dC primer can be added, annuals to this poly G strand and therefore a short double stranded section of DNA made where DNA polymerase can attach and form second DNA strand from cDNA
Before running a PCR, what kind of ends do you want your strands of DNA and primers attached to have?
You want sticky ends that will anneal easily, as at the end of the PCR, they need to anneal with the plasmid vector.
you can add a restriction site into the primer which will allow you to use restriction enzymes that cut sticky ends; more efficient
What does Taq polymerase do?
Adds an extra A onto the ends of fragments which creates and overhang, and therefore sticky ends which are easier to anneal
What is transformation?
Uptake of free DNA by bacteria.
Cells mixed with free DNA and DNA is simply taken up
(in animal cells, this is called transfection)
What is transduction?
DNA is incorporated into a virus and then transferred
What is conjugation?
Transfer of DNA through cell-cell contact
Cell cell contact may be made through Pili
Eg plasmids being passed round a bacterial population; most common way of getting antibiotic resistance.
What does is mean when bacteria are “competent”?
When they take up free DNA (ie are transformed) naturally, ie they’ll actively take it up themselves.
Streptococcus and bacillus species can do this
What happens when bacteria aren’t naturally competent?
A bacteria that isn’t naturally competent is e coli
It needs help to take up free DNA (eg plasmids)
You can mix it with CaCl2 and heat shock, which will form pores in its surface.
You could also use electro poration! Creates pores on cell surface using a very high voltage
Transfection, ie DNA being taken up into eukaryotes cells (like mammalian cells) does not occur naturally. What methods can be used to get DNA into these cells?
DNA incorporated into liposomes
Microinjection of DNA (just injecting DNA into cell )
Biolistic particle delivery (DNA coated in metals,gun shoots metes into cells, gene gun )
Also by electro poration (using high voltage to form pores)
What makes an ideal host to clone DNA in?
Non pathogenic Grows on a inexpensive media Will replicate Will take up DNA easily (eg competent bacteria) Allows high levels of gene expression Genetically stable
When we’re making pharmaceuticals that require proteins to be made, we want to make a large number of proteins. We do this by optimising _____&_____ of a gene.
Optimising transcription and translation of a gene.
Eg we could optimise transcription by using a strong promoter.
What system is good for making proteins for therapies?
The T7 RNA polymerase expression system
Polymerase in E coli transcribes T7 polymerase gene, making T7 polymerase. IPTG stimulates this.
T7 polymerase can then transcribe the target gene on a PET vector That is inserted into the E coli cell. IPTG stimulates this.
This results in lots of protein being produced, as you’re making more of the enzymes that are needed to make the proteins.
Good for making proteins for therapies as you get a lot of protein produced!
In the T7 polymerase expression system, when do you get over production of the protein, ie what TRIGGERS it?!
When you add IPTG you can get over produced protein, as IPTG switched on the expression of this protein and stimulates its transcription.
No IPTG; under production of T7 RNA pol
IPTG: over production of T7 RNA pol
You can visualise if you have had over production or under production on an SDS page plate
Three examples of therapeutic proteins?
Insulin
Trastuzumab to treat breast cancer
IL-2 to treat cancer
What is production method 1 for insulin?
Start with Seperate plasmids; 1 contains gene for chain A and galactosidase, other contains gene for chain B and galactosidase.
Insert each plasmid into Seperate E coli cell
Allow the proteins to be produced from the genes
Seperate and purify these proteins
Combine the A and B chains, they bind by disulfide bonds
Forms active insulin
What is production method 2 of insulin?
Start with proinsulin mRNA
Transcribe cDNA using reverse transcriptase
Fuse cDNA with B-galactosidase (so we can detect which cells take up the insulin DNA When we transform the cells)
Transform the cDNA into E coli
Wait for proteins to be made
Once made, cleave off B-galactosidase using CnBr
Cleave the pro insulins extra chain off using an enzymes
Left with INSULIN!
Why do we add beta galactosidase to the insulin in production methods 1 and 2?
This is so that we can detect which cells have taken up the insulin gene, as beta galactosidase attaches to the insulin A or B chain, and when add X-gal a blue substrate forms, so we can see which cells have taken up the insulin gene and beta galactosidase gene.
How do we cleave off beta galactosidase off our A and B chains?
Using CnBr
It cleaves after the Met residue where beta galactosidase attached to the A/B chain
What methods production of insulin method 3 similar to? What is different?
Similar to method 2
But instead of B-galactosidase we use B-lactamase, and it’s done in the peri plasm so more stable a proteins in periplasm don’t cleave insulin like they do in cytoplasm in methods1 & 2
What is production method 3?
Copy pro insulin mRNA into cDNA
Fuse cDNA with B-Lactamase
Transform E coli
Wait for proteins to be made in periplasm
Extract proteins from periplasm and purify
Convert pro insulin to insulin by cleaving off extra chain
Forms active insulin in periplasm
Cloning of factor VIII a protein used to treat heamophilia……
DNA library made in phage (a vector)
Gene for VIII taken from library, and is cloned in another vector (plasmid, pBR322 Ori)
Plasmid then cloned in hamster
Gene for factor 8 amplified using methotrexate
Cloning/ making this protein is carried out in MAMMALIAN cells as its large and glycosylated!!
What enzyme forms daptomycin?
It’s produced naturally by bacteria by NON-RIBOSOMAL-PEPTIDE SYNTHETASE
This enzyme is coded for by 3 genes: DptA DptBC DptD
What’s the main concept of combinational engineering?
We can use DNA technology to swap genes round to make different METABOLIC PATHWAYS
What host organisms are preferred for small proteins that don’t need to be post translationally modified?
Bacteria/ yeast preferred
Remember Bacteria won’t carry out post translational modifications, yeast can do but incorrectly
Insulin is made/ cloned in ______.
Factor 8 is made/cloned in ______.
Insulin in bacterial cells- E coli!
Factor 8 in mammalian cells– Chinese hamsters! this is because it’s large and glycosylated
