Genetic Circuits and Optimising Recombinant Expression

Question 1

What is the flexibility of the double stranded duplex a result of?

Answer 1

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.

Question 2

What is the mechanism of the lac repressor?

Answer 2

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.

Question 3

How can the DNA code itself control transcription?

Answer 3

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.

Question 4

What is codon preference, and why does it exist?

Answer 4

Many prokaryotes prefer to use a reduced set of codons within the ‘universal’ code.

Question 5

Why does Met have only one codon?

Answer 5

to reduce the likelihood of mutations that cause early starting proteins

Question 6

What are the advantages of having a codon bias?

Answer 6

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. .

Question 7

Why does codon bias prevent viral transcription?

Answer 7

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.

Question 8

How do viruses combat codon bias?

Answer 8

by encoding their own tRNA within their genome to ensure translation of their mRNA.

Question 9

How do ribosomes bind to an mRNA in prokaryotes?

Answer 9

They bind to the shine-dalgarno sequence (AGGAGGU) by their 16S subunit, which then recruits the 50S subunit.

Question 10

How can alterations in the S-D sequence regulate translation?

Answer 10

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.

Question 11

What is the size of the gap between ribosomes in polysomes?

Answer 11

minimum of 35bp

Question 12

How can bacteria be made competent and how do these methods work?

Answer 12

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.

Question 13

What is the size range of articial plasmids, and why?

Answer 13

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.

Question 14

What are plasmids within a bacterium referred to?

Answer 14

Plasmids within a bacteria are referred to as episomes, and the features of a plasmid are those that are needed for episomal independence.

Question 15

What must a plasmid contain for episomal independence?

Answer 15

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.

Question 16

What must plasmid cistrons be engineered with?

Answer 16

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).

Question 17

What is a trc promoter?

Answer 17

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.

Question 18

Why is the copy number of a recombinant plasmid important?

Answer 18

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.

Question 19

What is a replicon?

Answer 19

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.

Question 20

What plasmid is often used as a model replicative regulation system?

Answer 20

ColE1

Question 21

What upregulates replication of ColE1?

Answer 21

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.

Question 22

How is replication repressed in ColE1?

Answer 22

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.

Question 23

How can the ColE1 copy number be increased?

Answer 23

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.

Question 24

What defines incompatibility groups?

Answer 24

The type of system used to control replication, eg ColE1/Rop, can define incompatibility groups for plasmids.

Question 25

Why might it be necessary to express multiple genes in the same organism simultaneuously?

Answer 25

perhaps because they interact to fold, one may be a chaperone or one may be being used to regulate the expression of the other.

Question 26

How can co-expression circuits be arranged?

Answer 26

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.

Question 27

How are polycistronic circuits arranged?

Answer 27

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.

Question 28

How are non-polycistronic cis-expression circuits arranged?

Answer 28

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.

Question 29

Which method of cistron arrangement is prefereable for cis-coexpression circuits?

Answer 29

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.

Question 30

Why are soluble proteins often used in fusion tags?

Answer 30

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.

Question 31

What is often used to separate a protein from a fusion tag?

Answer 31

protease recognition motifs, the most common which is the TEV Protease cleavage site.

Question 32

What is the TEV protease motif?

Answer 32

seven residues long (N-ENLYFQ*S-C)

Question 33

Why is TEV protease often used?

Answer 33

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.

Question 34

Which end of the protein must the fusion tag be if a TEV protease recognition motif is used?

Answer 34

the N-terminal end, in order to avoid leaving more residues behind that could disrupt activity.

Question 35

Why might a TEV linked fusion protein be totally removed?

Answer 35

Sone proteins cleave off the methionine left on the N-terminus after translation, which often happens when the second residue is small. Leaving a serine or glycine behind after the cleavage can mean that this is not interfered with and all evidence of the tag can be removed.

Question 36

What is the sequence of an N-terminal fusion tag?

Answer 36

ATG - Tag codons - protease site (opt) - ORF - STOP

Question 37

What is the sequence of a C-terminal fusion tag?

Answer 37

ATG - ORF - protease site - Tag codons - STOP

Question 38

What may commercially available bacterial strains possess?

Answer 38

They will have their genome published with the different heritable modifications listed. These modifications may be mutated or deleted genes or genes introduced by plasmid or transposon. The genes may be engineered to be different or the ‘helper plasmids’ may contain regulatory factors to act in trans, for gene regulation or to provide chaperones/obligate binding partners/DNA motifs.

Question 39

What are cloning strains?

Answer 39

These are used to amplify the genes into a large library without causing any expression of the foreign genes that might create a selection pressure for them to be lost. This allows very large amounts of the gene to be cloned. The strains used are said to be impaired in recombination, they do not recognise exogenous promoters and lack certain nucleases that might break up the gene inserts.

Question 40

What are expression strains?

Answer 40

These are used to allow for high levels of expression, so tend to have better tolerance for recombinant proteins. Unlike cloning strains these can recognise exogenous promoters, but may regulate them tightly to allow for control of expression. They also lack certain proteases that might break up the recombinant protein.

Question 41

What is the T7 recombinant expression system?

Answer 41

This uses several elements from T7 genes, both inserted into the genome and in plasmids, in order to express recombinant genes in E. coli.

Question 42

What is a DE3 lysogen?

Answer 42

The T7 RNAP gene, modified for PLacUV5 expression control, is inserted into the bacterial chromosome via a lambda prophage. This bacteria is then called the DE3 Lysogen.

Question 43

What elements of the T7 recombinant expression system are not inserted into the bacterial genome?

Answer 43

- The lysozyme cistron (T7 gene 3.5) - The target ORF controlled by the T7 gene 10 promoter - The gene 10 stem-loop transcription terminator. The expression plasmid may use a PLacUV5 modification to the T7 promoter to allow for expression control; 0.5-2% glucose inhibition and 0.1-1mM IPTG induction via action upon the lac repressor.

Question 44

Why is the T7 recombinant expression system useful?

Answer 44

the T7 RNAP can transcribe genes from T7 promoter incredibly quickly, and the T7 Φ 10 promoter in particular gives very strong binding. The system can give around 35x higher transcription rates than other, non-viral expression systems meaning that the target mRNA transcript can account for up to 70% of the transcriptome.

Question 45

What is one reason the T7 recombinant expression system gives such high transcription rates?

Answer 45

The T7 RNAP does not recognise the prokaryotic bipartite (-10 and -35) promoters, only the 10-11 nucleotide viral promoters.

Question 46

What is the purpose of the lysozyme in the T7 recombinant expression system?

Answer 46

This is an alternative form of regulation to using Lac modifications – it binds the T7 RNAP in a 1:1 ratio, inhibiting it and so blocking transcription. Low level constitutive expression of lysozyme is used in conjunction with PLacUV5 control to prevent ‘leakage’ of the lac-controlled gene due to trace amounts of lactose. This can also be fixed by culturing with 0.5-2% (w/v) glucose or increased lac repressor.

Question 47

What is the problem with using lysozyme in the T7 recombinant expression system?

Answer 47

The activity of lysozyme is to disrupt the membrane of the host cell, killing it. This causes a lot of problems as when one cell bursts it releases lysozyme which kills nearby cells causing a chain reaction that leaves very little population.

Question 48

How can the lysozyme toxicity issue be resolved in the T7 recombinant expression system?

Answer 48

A mutant lysozyme called lysY is used, in which an active site lysine is mutated to tyrosine (K128Y), neutralising the function of the enzyme while preserving its structure and so inhibitory abilities.

Question 49

Which strain is most often used for the T7 recombinant expression system sans lysozyme toxicity?

Answer 49

The BL21 [DE3] strain is commonly used for that, as not only is it a DE3 lysogen but also has the Ion and OmpT proteases knocked out to prevent protein degradation. Lowering the growth temperature can also minimise proteolytic degredation.

Question 50

What parts of a recombinant expression system can be optimised?

Answer 50

Recombinant DNA Bacterial strain Pre-culture/Inoculum Culture conditions

Question 51

What issue is raised by long term recombinant experiments?

Answer 51

When it is necessary to perform multiple experiments on a transformed bacteria, the host can be freshly transformed each time but this is a costly and time consuming way of overcoming the loss of expression due to natural selection.

Question 52

How can cells be archived so as not to have to re-transform them many times?

Answer 52

To get around this the actively dividing cells can be archived by freezing them at -80°C, and frozen colonies scraped off for use in culturing. 8% (v/v) glycerol can be used as a cryoprotectant, but this technique is still not suitable for bacteria that are expressing toxic proteins.

Question 53

What culture conditions can be optimised, and how?

Answer 53

• Medium o Rich or minimal o Addition of particular metabolites • Temperature o Ranges from 15-37°C • Aeration and agitation o Shake flask or fermentor  Geometry of container can be important • Duration o Of growth before induction or time after induction until harvesting • Induction o Some media formulations enable auto-induction, useful if titration of inducer is impossible • Harvesting o Continuous or Batch culture

Question 54

What are the standard conditions for E. coli batch culture?

Answer 54

• 2L Erlenmeyer flask • Luria Bertani (LB) medium + antibiotic for screening • 37°C until induction, then dependent on protein • 1:5 (v/v) aeration • 200rpm agitation • IPTG induction in mid-late log phase o Cells sampled at 2 and 20 hours minimum, maybe also 1, 4, 8 and 16.

Question 55

What is the standard E. coli batch culture used for?

Answer 55

To easily test for recombinant expression.

Question 56

What modifications may have to be made on the standard E. coli batch culture protocol depending on the protein?

Answer 56

supplementing the culture with metal ions or amino acids, testing for leaky expression and any effect it may be having and comparison between auto-induction media and LB.

Question 57

When a test for recombinant proteins obtains unstable, insoluble or incorrectly folded protein or no protein at all due to proteolysis, how can conditions be changed to minimise these issues?

Answer 57

This can be minimised by lowering the growth temperature, co-expression with chaperones or partner molecules or mutation of the protein. (Bi)Terminal mutation is a particularly common method for obtaining viable recombinant proteins.

Question 58

What issue is presented by the cytoplasm of E. coli, and how can this be overcome?

Answer 58

Many proteins will not form in the cytoplasm of E. coli. This may be because of the reducing environment, in which case the protein can be tagged for export into the less reducing periplasm. Also, proteins that require PTMs to fold will not find them here.

Question 59

Where else in the E. coli may a protein be targeted to account for its properties?

Answer 59

Membrane proteins may need to be inserted into the plasma membrane to fold. High toxicity of the protein may mean that it has to be secreted from the cell to prevent immediate death.

Question 60

What is an alternative organism used to overcome E. coli expression issues?

Answer 60

B. subtilis

Question 61

What is the purpose of combinatorial domain hunting?

Answer 61

To get around issues of folding more complex proteins, shotgun fragmentation of the gene using an endonuclease is used to introduce only single domains into a plasmid for transformation and expression, thus allowing for isolated study. This allows the different domains and fragments to be screened for solubility, activity, correct folding etc simultaneously, which can also identify which part of the protein is unable to fold in this expression system.

Question 62

How is CDH performed?

Answer 62

- Amplification of gene by PCR - Uracil DNA glycosylase treatment excises all Uracil bases - AP endonuclease cleaves areas of base excision, shotgunning the gene - TOPO cloning - Create Shotgun Cloned Expression Library Construct - High throughput screens for recombinance

Question 63

What is CDH2?

Answer 63

This can be extended to CDH2 screening, in which the protein fragments are combined randomly in every different combination to see which interact to fold together. This is used to identify the core interactions of protein complexes.

Question 64

What different versions of CDH2 exist?

Answer 64

This technique may have a huge number of permutations if there are many fragments. There are two different typed of CDH2, All vs All and One vs All.

Question 65

What is One vs All CDH2?

Answer 65

One vs all is used to reduce the number of screens by testing one fragment against all others and examining the results, but this does require the one fragment to be stable independently.

Question 66

What is all vs all CDH2?

Answer 66

All vs all pairs every different domain to each other, which is useful for finding structures which completely rely on each other for formation.

Question 67

What are cell-free expression systems?

Answer 67

This is the use of extracts from living cells that make up everything needed for translation (and sometimes also transcription) to produce in vitro expression systems.

Question 68

What are the two components of cell-free expression systems?

Answer 68

The permanent and reusable ‘reaction components’ and the base materials they will convert into protein; the ‘feeding buffer’.

Question 69

What are the reaction components of of cell-free expression systems?

Answer 69

* Ribosomes * Translation factors * tRNA * Aminoacyl tRNA synthetases * mRNA * RNAPs + DNA (if transcribing too)

Question 70

What is the feeding buffer of cell-free expression systems?

Answer 70

* Amino acids * Energy components (ATP + GTP) * Cofactors and accessory reagents * NTPs (if transcribing)

Question 71

What are the main types of cell-free expression systems?

Answer 71

Two batch variants: Basic batch culture continuous exchange Two continuous variants: Continous flow Bilayer continuous

Question 72

What is Basic batch culture?

Answer 72

Basic batch culture can be used with all the components in a single receptacle, and the product then being purified.

Question 73

What is continuous exchange?

Answer 73

a dialysis membrane divides the container allowing for more feeding buffer to be added to the other compartment but the protein product cannot cross into the other compartment. This has higher yield than normal batch culture.

Question 74

What is continuous flow?

Answer 74

Continous flow involves the feeding buffer to be pumped into the reaction cubicle (containing the reaction components) and the resulting mix to be pumped out through a ultrafiltration membrane, causing the feeding buffer and protein product to be removed but leaving the reaction components.

Question 75

What is bilayer continuous exchange?

Answer 75

Bilayer continuous systems can also be used which rely on diffusion of the feeding buffer and proteins only through an interphase filter. This differs from normal continous exchange in that the proteins can be constantly removed.

Question 76

What are the advantages of cell-free expression?

Answer 76

Cell free expression avoids issues of toxicity and the difficulties in expressing things in a complex environment – i.e. there are no proteases and the redox state/pH can be carefully controlled. There are also few contaminating protein products to be purified out, and limits the need to denaturing/renaturation. Unlike cell expression this allows for the possibility of adding PTMs to the proteins, and can be optimised so that the protein forms the correct disulphide bonds.

Question 77

What are the disadvantages of cell-free expression?

Answer 77

Cell free expression is far more expensive per unit of target protein than expression in a host cell. It can also be very tricky and difficult to optimise.

Question 78

What is synthetic biology?

Answer 78

The design of new kinds of biological entities using an engineering ethos of modular construction, part standardisation and performance and tolerance data.

Question 79

What are some aims of and applications for synthetic biology?

Answer 79

Synthetic Microorganisms (novel genomes) - use in environmental and industrial science, food, medicine fuel etc. New genetic codes/precursors for GM firewalls Novel enzymes/pathways (same applications as synth. microbes) Nano-scale devices from microbes & macromolecules - DNA/protein sensors, circuitry and computing.

Question 80

What progress has been made on synthetic microorganisms?

Answer 80

Recreating bacteriophage T7 genome from scratch Synthetic genomes of M. genitalium and M. mycoides/M. capricolum

Question 81

What progress has been made on GM firewalls?

Answer 81

Four-letter genomic code system produced with quadruplet decoding ribosome xenobiological artificial bases using different sugars produced, eg threose (TNA), hexose (HNA) and glycol (GNA)

Question 82

What progress has been made on environmental uses??

Answer 82

E. coli used to produce electricity E. coli used to make biofuels

Question 83

What progress has been made on macromolecular circuitry?

Answer 83

Evaporative, lithography free printing of DNA wires