Biotechnology Flashcards
What are the two culture methods?
Batch and continuous.
How do either of the methods differ?
Batch is a closed culture method where the products are removed at the end of the culture. Continuous culture has a constant inflow and outflow of substances. The bacteria is maintained in a homogenous grwoth condition.
What is the problem with producing eukaryotic proteins in prokaryotes?
There are major differences between prokaryotes and eukaryotes such as the regulatory sequences, introns, post-transcriptional processing/post-translational processing - so it is not as simple as it may seem!
What is a promoter?
Where transcription of a gene starts - RNA polymerase II binds.
What is a terminator?
Where transcription stops.
What is a ribosome binding site?
Where ribosomes attach to mRNA.
How do promoters/terminators and ribosome binding sites differ between eukaryotes and prokaryotes?
They are all different, meaning eukaryotic genes cannot work in a prokaryotic background.
How can the differences between prokaryotic and eukaryotic genes be solved?
Use a prokaryotic promoter and signals to drive the eukaryotic gene, such as special expression vectors that are already in the vector.
What does using cDNA instead of the genomic clone mean?
It eliminates the problems from introns in eularyotic genes, but E.coli does not always process proteins as eukaryotic cells do.
What do E.coli expression vectors contain?
E.coli control sequences such as promoters, ribosome binding sites, restriction sites and terminators. The restriction site is located between the promoter and terminator.
How do strong promoters and weak promoters differ?
Strong promoters sustain a high rate of transcription and are used for products that are needed in large amounts, whereas weak promoters are relatively inefficient and useful if your product is toxic at higher levels.
How can more sophisticated promoters work?
They can switch the gene on and off as convenient as some foreign proteins that need to be replicated may be harmful to the E.coli. This means large amounts can still be produced even if the E.coli cells die.
What is an example of a more sophisticated regulatable promoter?
The lacZ promoter that can be activated with IPTG (an artificial inducer) to switch on the lacZ gene.
How can the Trp promoter be used?
The TrpA gene is involved in the biosynthesis of trptophan. If 3-beta-indoleacrylic acid is added it will be switched on and transcription will occur. Adding tryptophan will repress it as there is a negative feedback loop to prevent overproduction of tryptophan.
How does the lambdaP1 promoter work?
It is temperature sensitive. Temperatures below 30 degrees allow no transcription.
What is alternative method to solve the problem of the differences between prokaryotic and eukaryotic cells?
Express the eukaryotic genes in eukaryotes instead.
What are the benefits of using yeast to express eukaryotic proteins?
It is a well characterised system, it has a range of useful promoters, any differences in regulatory sequences from mammals is not a problem.
What are the problems of using yeast to express eukaryotic genes?
Hyperglycosylation - some amino acids such as asparagine in humans is glycosylated, but in yeast there will be an excess of glycosylation. The secretion of proteins from yeast is also not efficient.
What is another method, aside from yeast, to express eukaryotic proteins?
Expressing the proteins in animal cells.
What is the method of expressing eukaryotic proteins on animal cells?
Cell culture - growing them on surfaces such as internal plates or inert particles such as cellulose beads.There is much slower growth than with microorganisms.
How can they be grown on mammalian cells?
Using expression vectors with strong promoters, such as from SV40.
How can they be grown on insect cells?
Expression system based on baculoviruses - the protein accumulates in inclusion bodies to high levels.
What is another way to express eukaryotic proteins?
Expressing them in whole organisms (transgenics).
How can transgenic animals be achieved?
Gene injection into the somatic nucleus. The nucleus is transferred to an oocyte with no nucleus and is then implanted into a foster mother to form an ovum. This is not a simple procedure.
