L2 Recombinant tech 2 Flashcards
(17 cards)
Generating recombinant DNA - 3
- Combining DNA from a source with a vector, to produce recombinant DNA
- Many proprietary systems available for generating rDNA (easy but expensive)
- Traditionally rDNA is made with specific enzymes
A) cutting: restriction enzymes
B) pasting: ligase
Restriction enzymes for cloning - 6
- Recognise palindromic sequences: restriction sites
- Restriction sites for cloning usually are 4 or 6 nucleotides
- Cut both DNA strands, creating sticky or blunt ends
- When cleaved produces sticky ends due to paired bases facing each other
- Blunt ends are more difficult to stick together
- e.g. CGGCCG reverse is CGGCCG
When cut DNA fragment, to determine it’s the right size Agarose Gel Electrophoresis - 4
When cut DNA fragment, to determine it’s the right size Agarose Gel Electrophoresis - 4
1. Load on agarose gel
2. Run for an hour at negative charge
3. Stain with fluorescent dye to visualize under UV light
4. Size can be calculated from position of MW markers
DNA Ligase - 4
- ATP-dependent enzyme that links DNA strands
- Plays a role in DNA repair & replication
- Can ligate compatible sticky ends, as well as blunt ends
- Ligation of sticky ends is more efficient than ligation of blunt ends
Vector DNA cut with restriction
Complimentary sticky ends ligated together
Important regions for Plasmids in cloning - 2
Important regions for cloning:
1. Selection marker (genes for antibiotic-resistance or growth on specific media)
2. Region where DNA can be inserted
Cloning workflow - 5
- Vector prepared using restriction digest, then purified
- PCR of insert, followed by restriction digest, then purification
- Vector & insert combined using ligase
- Prepare competent cells (heat pulse on E.coli)
- Plate cells on colonies, these are screened using Blue white-screening
Blue white screening - 5
- Plasmid contains lacZ gene, which encodes the enzyme β-galactosidase (degrades lactose)
- If lacZ gene is intact: β-gal active and converts artificial substrate X-Gal into blue dye
- If DNA insert disrupts lacZ gene: β-gal inactive no conversion of X-Gal into blue color
- Colonies with intact lacZ (no insert) are blue
- Colonies with inactive lacZ (with insert) are white
Other vectors: A or B
A) λ cloning vectors
based on bacteriophage lambda
can accommodate larger inserts
B) Artificial chromosomes
constructs based on bacterial or yeast DNA
Can contain very large inserts
For cloning large genes, genome mapping and genome sequencing
Hosts for cloning and expression - 5
- Grows rapidly in inexpensive medium
- Non-pathogenic
- Is genetically stable
- Has many tools for genetic manipulation
- Allows high level of expression of genes
Insulin - 3
- Hormone produced in pancreas
- Controls blood sugar levels
- Faulty production or inefficient utilisation leads to diabetes
Production of recombinant insulin - 4
- short peptides such as insulin are not very stable (due to degradation) in cytoplasm of E. coli
- peptides can be stabilised by fusion to a large protein
- sequence of insulin can be modified if desired
4.e.g. amino acid changes in insulin can be used to make insulin fast- or slow acting
Recombinant human insulin - 4
- Clone insulin A & B chains separately in E. coli, as fusions with gene encoding β-galactosidase
- Purify fusion proteins & cleave off β-gal
- Combine A & B chains & refold in oxidising conditions in vitro
- oxidising conditions are required to form disulphide bonds for stability & activity of the protein
Modulating insulin-release profile - 3
- Mix with protein to slow release e.g. NPH
- Introduce amino acid changes
- Chemical modification
Lispro - 3
Lispro:
1. reversal of Lys/Pro in B chain
2. Forms dimers very inefficiently
3. Rapid release
Glargine - 3
Glargine:
1. One deletion and 3 additions: pI 5.4 to 6.7
2. pI close to neutral pH: soluble at acidic pH but precipitates when injected subcutaneously – released from precipitate
3. Slow release
Factor VIII - 5
- Essential blood clotting factor
- Used for treatment of haemophilia
- Very large protein of 2332 AA
- Largest recombinant protein that is used commercially
- Glycosylated
Cloning of Factor VIII - 4
- Very large gene with several introns - requires copies to be made from mRNA
- Initial cloning was done in E. coli
- Plasmid containing F8 gene used to transfect mammalian cell lines
- Plasmid integrates in genome; number of copies amplified, & cell line with highest number of copies used for production
