Genetic Engineering & Microbes Flashcards
Biotechnology definition
Use of living organisms for industrial/commercial applications
GMO definition
An organism whose genome has been altered
What does genetic engineering allow the expression of
Eukaryotic genes in prokaryotes e.g: insulin
How is the expression of eukaryotic genes in prokaryotes achieved
By cloning the gene via mRNA
What components does the process of expressing a recombinant mammalian protein in an expression system (5)
- Identification of gene that encodes the protein of interest
- Generation of cDNA from the rRNA
- Selection of expression vector to insert gene sequence
- Selection of suitable system that can express vector
- Appropriate screening & scaling up methods
Steps of getting from gene to protein
-Transcription (RNA synthesis)
- RNA processing (remove introns, and exons spliced together)
- Translation (protein synthesis)
What is reverse transcription
mRNA -> cDNA
Steps in reverse transcription
- Addition of primer & reverse transcriptase
- Reverse transcription to form single stranded cDNA
- Addition of RNase H
- Addition of primer specific to 5’ end & Taq polymerase
- Have double stranded cDNA
Steps of E. coli cells taking up plasmid DNA of interest
- Transformation of competent E. coli with genes of interest
- Selection & expansion of positive clones
- Isolation of plasmid/expression vector
- Isolation of protein of interest
What problems is protein synthesis in a foreign host subject to facing
- Degradation by intracellular proteases
- Toxicity to prokaryotic host
- Formation of inclusion bodies
In the expression vector, where does RNA polymerase bind & what does it do
Binds at the promoter & synthesises the gene
In an expression vector, what does the origin of replication allow for
Allows the plasmid to replicate
Steps in production of recombinant bovine somatotrophin
- Starts with bovine somatotropin mRNA from cow
- Convert BST mRNA to cDNA using reverse transcriptase
- Insert into expression vector
- Transform vector into E.coli cells
- Produce recombinant BST commercially
- Inject rBST into cow to increase milk yield
Some therapeutic products made from genetic engineering
- Erythropoietin to treat certain anaemias
- Factors 7, 8, 9 to promote blood clotting
- FSH for tx of reproductive disorders
- Insulin for tx of diabetes
- Somatotropin (growth hormone) for tx of growth abnormalities
- Colony stimulating factor for tx of infections & cancers
- Lysozyme for anti-inflammatory
