Recombinant DNA Technologies Flashcards
Recombinant DNA
Join sections of DNA together
Inserted into another organism to produce protein
Manipulation of recombinant DNA
Plasmid
Restriction enzymes
DNA ligases
Transformation (amplify plasmids)
Plasmid
Circular piece dsDNA
Origin of replication
Antibiotic resistance gene
Promoter
Origin of replication
Initiation of replication using host DNA poly
Antibiotic resistance gene
Selection of cells with plasmid
Promoter
Drives expression of gene (with transcription factors)
Correct type with a certain type of cell used (ie. prokaryotic needs protomer used in prokaryotic cells)
Restriction enzymes
Cut dsDNA at specific sequence
DNA ligase
Phosphodiester to bond with DNA backbone via sticky ends
Transformation and replication of plasmid
Transfer plasmid into bacteria - heat shock
Select bacteria by antibiotics
Bacteria replicate with plasmid
Not using bacteria to produce recombinant DNA
Same but
3
Transfrom into bacteria (increase plasmid) then transfer into eukaryote
Universal genetic code
All organisms “read” same genetic code
Can produce same protein with human gene in bacteria
Problem with universal genetic code
Prokaryotic genes can’t process eukaryotic introns
Steps to produce recombinant protein
1) Isolate
2) Clone plasmid
3) Transform bacteria
4) Grow cells with protein of interest
5) Purify
Insulin gene of interest
cDNA = no introns
Only A & B held by disulfide bonds
Must be made in separate bacteria as pre-proprotein (doesn’t occur in bacteria)
Insulin purify
IacZ = 2 selector (removed later)
Fuse protein
Cyanogen bromide cleave A&B
Mix = functional insulin
Insulin in normal human
Preproinsulin - roughER -> proinsulin 3 disulfide bonds b/w a and b
proinsulin - golgi -> insulin c removed
Sources of therapertic proteins
Non human
Prokaryates
Human
Non-human therapeutic protein eg and problems
Pig, cows
E.coli
Immue response, incorrect structure = incorrect function
Human therapeutic protein eg and problems
Blood Potentially infectious (blood borne pathogens), low yield, tissue source
Advantages of prokaryotic system
High yield
Low cost
Pathogen free
Disadvantages of prokaryotic system
Proteins partially folded
Inability to form post-translational modifications
Advantages of mammalian cells
Pre-proprotein
Processed efficiently and purification easier
Disdvantages of mammalian cells
Expensive
EPO
Chinese Hamster Ovary cells
Glycosylation = PTM
-> human and recombinant look different after glycoslation
Pharming
Using whole animals
Anti-thrombin
Decrease = spontaneous blood clots
Control AT production
Anti-thrombin pharming??
Transgenic goat
Promoter that control gene ????
Milk specific promotor (easy, doesn’t harm goat)
Anti-thrombin problem
Can’t use prokaryotes as PTMs essential (gamma – carboxylation)
Designing new proteins
Create random mutation = regenerate PCR
Replicate in bacteria and analyse
Gene therapy
Plasmid target specific cells using viral vectors
Stable/permanent as it intergrates into host cell chromosome
Type 1 diabetes
Beta cells destroyed - autoimmune disorder
Plasmid + pre-proinsulin cDNA make liver cells insulin producing
Gene editing
CRISPR-CAs
