Genetic Circuits and Optimising Recombinant Expression Flashcards
What is the flexibility of the double stranded duplex a result of?
The variety of conformations adjacent base pairs can have relative to one another.
It is this that allows DNA to curve, coil and supercoil. Some curvature is even needed to form circularised plasmids.
What is the mechanism of the lac repressor?
It forms dimers on an operator before and one after the promoter. These then come together to form a tetramer and coil the DNA up between them in such a dramatic way that RNAPs cannot bind to the promoter.
Even if a polymerase complex could assemble it would be unable to elongate.
How can the DNA code itself control transcription?
By changing the speed of the RNAP by changing the ratio of AT to GC base pairings thus making the DNA easier or harder to separate.
What is codon preference, and why does it exist?
Many prokaryotes prefer to use a reduced set of codons within the ‘universal’ code.
Why does Met have only one codon?
to reduce the likelihood of mutations that cause early starting proteins
What are the advantages of having a codon bias?
It reduces the number of tRNAs that it is necessary to produce in abundance but also is an attempt to thwart the translation of viral proteins that use the rare codons. .
Why does codon bias prevent viral transcription?
it reduces the processivity of the ribosome by causing it to pause while searching for the correct tRNA anticodon to complete the next step in the chain. When paused it is far more likely that the ribosome will dissociate from the mRNA, ceasing translation. However this is an inexact method, quite often a suitably similar tRNA can be found or it may be the rare case in which that tRNA is present.
How do viruses combat codon bias?
by encoding their own tRNA within their genome to ensure translation of their mRNA.
How do ribosomes bind to an mRNA in prokaryotes?
They bind to the shine-dalgarno sequence (AGGAGGU) by their 16S subunit, which then recruits the 50S subunit.
How can alterations in the S-D sequence regulate translation?
Similar to the promoter this sequence can have small variations that reduce or increase affinity that increase the residence time – the time for which the 16S subunit is likely to remain bound before falling off, assuming that it is not able to recruit the 50S subunit.
What is the size of the gap between ribosomes in polysomes?
minimum of 35bp
How can bacteria be made competent and how do these methods work?
This can be done chemically (often using calcium ions, the mechanism of which is unclear) or by electroporation which is thought to work by disrupting the membrane.
What is the size range of articial plasmids, and why?
between 2 and 10kb.
Larger plasmids are difficult to work with as they are liable to shear (and potentially linearise), preventing proper transformation and expression.
What are plasmids within a bacterium referred to?
Plasmids within a bacteria are referred to as episomes, and the features of a plasmid are those that are needed for episomal independence.
What must a plasmid contain for episomal independence?
Each plasmid must have a selection marker and a replicon (i.e. ORI and associated genes). Plasmids can have any number of cistrons (complete genes) limited only by the size limit of the vector.
What must plasmid cistrons be engineered with?
each cistron must contain a:
- promoter,
- operator (to allow for repression/induction),
- ribosome binding sequence (S-D),
- a start codon (possibly with space for fusion tag/protease site)
- a stop codon plus termination sequence (eg hairpin loop).
What is a trc promoter?
By combining the -10 promoter region and operators of the Lac UV5 gene and the -35 promoter region of the Trp gene (which provides strong RNAP binding) a hybrid trc promoter can be made which is stronger than either of them and also inducible.
Why is the copy number of a recombinant plasmid important?
If the gene is constitutively expressed the copy number will be proportional to the level of expression.
If the gene product is toxic and is going to be harvested using an induced burst of high expression when the bacterial population is established, a high copy number is essential for ensuring that each cell produces a large amount of the gene before dying.
What is a replicon?
A replicon is the sum of the DNA replicated from one origin. Chromosomes usually have multiple ori but plasmids only have one, so each is a replicon.
What plasmid is often used as a model replicative regulation system?
ColE1
What upregulates replication of ColE1?
ColE1 transcribes a 555 nucleotide RNA called RNA II to act as a primer at the ori, thus increasing the rate of replication. To be able to do this it must be processed by RNase H, which cleaves it to leave a properly positioned 3’-OH for DNA Pol to recognise.
How is replication repressed in ColE1?
A partial antisense of RNA II can also be transcribed – called RNA I – that prevents RNA II from being activated by RNase H.
This inhibition of RNA II is accelerated by the presence of a gene transcribed on the plasmid called Rop (repressor of primer), a small dimeric protein that aids the RNA duplex formation and so inhibits replication.
How can the ColE1 copy number be increased?
By knocking out the Rop gene you can limit the inhibitory action of RNA I, meaning that the plasmid is permanently acting as though there are too few copies and so continuously replicates.
What defines incompatibility groups?
The type of system used to control replication, eg ColE1/Rop, can define incompatibility groups for plasmids.
Why might it be necessary to express multiple genes in the same organism simultaneuously?
perhaps because they interact to fold, one may be a chaperone or one may be being used to regulate the expression of the other.
How can co-expression circuits be arranged?
These can be arranged in cis or in trans by having the cistrons on the same or on different plasmids, or any combination therein.
When two proteins need to be expressed from one plasmid they can be arranged in a polycistronic way or as separate cistrons.
How are polycistronic circuits arranged?
Polycistronic arrangements use a single promoter with a long transcript, with a Shine-Dalgarno sequence before each open reading frame (with small gaps in between all sequences) and ending in a single termination site.
How are non-polycistronic cis-expression circuits arranged?
The two ORFs can be controlled using discrete promoters to produce two mRNA transcripts, meaning that each ORF requires its own promoter, SD sequence and termination site.
Which method of cistron arrangement is prefereable for cis-coexpression circuits?
Non-polycistronic, as some SD sequences within long mRNAs are often not recognised by the 16S ribosome so there can be issues with translation of the second ORF. The reasons for this are unclear, but may be due to formation of local secondary structure in the mRNA.
This does sometimes work to the advantage of the researcher when a Shine-Dalgarno sequence happens to appear in a eukaryotic gene that is being expressed in a prokaryote, as this can cause translation to be framshifted and incomplete.
Why are soluble proteins often used in fusion tags?
Glutathione S Transferase and Thioredoxin A are commonly used as fusion proteins because they are incredibly soluble and so provide an unspecific scaffold for solubilising the target protein, reducing the level of precipitation.
What is often used to separate a protein from a fusion tag?
protease recognition motifs, the most common which is the TEV Protease cleavage site.
What is the TEV protease motif?
seven residues long (N-ENLYFQ*S-C)
Why is TEV protease often used?
It is not part of a common domain in eukaryotes, and the length of the recognition site means the TEV protease is highly unlikely to find an exposed motif and be able to cleave the target protein.
Also cleaves to leave only one residue behind.
