R.O Lecture 5 Flashcards
What validation methods can be used to determine if DNA cloning was successful?
1.Selectable markers
2.Blue/white colony screening
3.Restriction enzyme digest
4.Colony PCR
5.Sequencing
6.Recombinant protein detection and analysis
- Selectable Markers
Selectable markers are often antibiotic resistance genes. They help in artificial selection. They help in identifying transformed cells and selectively allowing their growth whereas eliminating non-transformants in cloning vector.
- What type of genes are used as selectable markers?
Can be antibiotic resistance genes e.g. ampr , tetr
Encode proteins that make the cell resistant to relevant antibiotics
ampr
ampr : encodes enzyme that hydrolyses active portion of ampicillin
tetr
tetr : encodes efflux pump that removes tetracycline from the cell
How do we know that the plasmid has been taken up by the cells? (selectable marker)
Select for transformed cells: Only bacteria that take up the plasmid with the selectable marker will be able to grow in the presence of the antibiotic
What method is used to insure that the “DNA of interest” has been succesfully inserted into the plasmid at the MCS?
Blue/white colony screening
What vector is blue/white colony screening used for?
Used in DNA cloning not expression plasmids.
What gene is used in Blue/White colony screening?
lacz gene
What enzyme does the lacZ gene encode for?
*MCS is located within the
lacZ gene on the plasmid
*lacZ gene encodes the β galactosidase enzyme;
under the control of the lac promoter
What does the βgalactosidase enzyme do?
*β galactosidase cleaves lactose into glucose and
galactose
*β-galactosidase is made up of two fragments, alpha and omega
When the two fragments are associated they form functional enzyme
*It Converts substrates such as X-gal (5-bromo-4-chloro-3-indolyl-[beta]-Dgalactopyranoside) a colourless modified galactose sugar into a blue coloured product
How are white colonies formed?
If a DNA insert is cloned into the MCS within the lacZ gene, the gene will be
disrupted, β-galactosidase will not be produced, and X-gal will not be
metabolised (white colonies).
IPTG
IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a molecular mimic of allolactose, a lactose
metabolite that triggers transcription of the lac operon: used to induce expression of inserted gene
What do blue and white colonies represent?
Blue colonies therefore show that they may contain a vector with an uninterrupted lacZα (therefore no insert), while white colonies, where X-gal is not hydrolyzed, indicate the presence of an insert in lacZα which disrupts the formation of an active β-galactosidase.
Diagnostic Restriction Enzyme Digest
Using restriction enzymes, can perform a diagnostic digest of plasmid to determine if DNA insert has been incorporated
Fragments generated will be different sizes depending on
whether the DNA insert is present or not
What does Diagnostic Restriction Enzyme Digest detect?
Analyse the digested plasmid on an agarose gel to determine if generated fragments are the correct size.
Can also determine if the DNA insert is in the correct
orientation in the MCS
What does Colony PCR Detect?
Detects presence/orientation of DNA insert using
primers designed to amplify a region of the
insert/plasmid
How is Colony PCR carried out?
- Transformed bacteria are spread on agar
plates to isolate individual colonies. - Primers are designed to anneal to the
insert/plasmid (green, blue, red arrows). - PCR is carried out using DNA from each colony
- PCR product is analysed by gel electrophoresis
to determine if it matches predicted size.
(look at slide 7 for diagram)
Sequencing
Sequencing the recombinant plasmid is the most accurate way to determine if insert has been incorporated correctly
Can determine if:
- the insert is in the correct orientation within the plasmid
- the inserted gene is in frame for correct translation
- the gene sequence is the same as reported in the genomic database
What are the methods for sequencing?
- Sanger sequencing (uses fluorescent dideoxynucleotides)
- Next-generation sequencing (for larger sequences)
What method is used to verify if the inserted “gene of interest” produce the expected protein product?
Recombinant protein production and analysis
Describe Recombinant protein production and analysis.
Following cloning of a gene into an expression plasmid and transformation
into a host system, protein expression can be induced and the protein of
interest can be purified and characterised.
Peptide tags (e.g. 6xHis Tag) or fusion
proteins (GST) can enable detection
and purification recombinant protein.
What are the steps in recombinant protein purification?
- Design expression plasmid, transform, select
- Grow culture of positive clone*, induce expression
- Lyse cells
- Isolate protein-containing fraction (centrifugation, protein solubilisation)
- Purify from contaminating proteins (Column Chromatography—collect fractions)
- Assess purity on SDS-PAGE (Detect with antibodies etc.)
What can be used to identify positve clones?
Verification methods discussed previously can be used to identify
positive clones containing the ‘gene of interest’
