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Nitric Oxide Sensing & The Hybrid Promoters | The Comparator Circuit | Theoretical Projects

PASCOE TO DO

SUMMARY OF WHAT WE'VE DONE HERE OVERALL WITH THE THEORETICAL PROJECTS; BASICALLY THAT WE'VE THOUGHT OF OTHER PROJECT IDEAS ETC.

multiplicative circuit

WHAT IT IS (CONCEPT EXPLANATION), MATHS FOR IT, RUSSELL TO PRODUCE GRAPHICS, THEORETICAL LABS

There are four central mathematical operations; subtraction, multiplication, addition and subtraction; the ability to do each author is in a cell unleashes massive potential for future applications.

A multiplier effect can be produced by using three loop system that causes attenuation in the tryptophan operon. The removal of the ribosome binding site up stream means the formation of the stem and loops are no longer dependent on the ribosome. Instead the team designed a synthetic gene that would produce an arm and a that would complimentary bind to cite one in their leader causing the sight three four loop to form and transcription to be attenuated. The probability of this happening is dependent on the concentration of the complimentary RNAs but also on the transcription initiation rate all the promoter creating a multiplier effect (where the rate of transcription initiation in the promoter is multiplied by a number between nought and one)

Multi-Sensor System

Any problems encountered again and again by synthetic biologists is that specific promoters do not exist for a particular ligand and it is very difficult to construct a transcription factor that is specific to the ligand required. There are however often broad spectrum (non-specific ) promoters that can be found for a particular ligand these promoters and their transcription factors will induce transcription initiation when exposed to (or not exposed to) this ligand but also when exposed to other similar ligands. Assuming competitive binding there is an interesting effect which can be exploited to give specific and accurate concentrations of each of the ligands which will bind to and that transcription factor. In its simplest form if there are two different transcription factors each of which will cause transcription when exposed to either or both of two different competitive ligands with different lead constructive active sites and then there will be a different bias in each active site to each ligand meaning any particular transcription rate in one of the promoters indicates any of a continuous range of ratios between the two different ligands (for example nitrates and nitrites) as seen on line 1 (figure 1). Because the two different construction factors have different binding efficiencies to the two different ligands the line of the other promoter will take a different angle (line 2) the point when these two lines cross gives the concentration of both ligands specifically even though the two different promoters are non-specific.

this effect can be modelled on more than just two substrates. Visually the system can be modelled with each concentration being a different access on a graph (which can soon become hyper dimensional) when you add a third ligand it can soon be seen that the two lines on figure 1 become two planes which intersect along one line (fig 2 ). this means that a third promoter and transcription factor are necessary (the same way that two pinpoint any single point in three-dimensional space it must be triangulated from three other points). Once the third promoter and transcription factor is added the three planes created intersect at a single point which gives the specific concentration of each of the ligands( fig 3 ) . When four ligands are used a hyper dimensional graph of four spatial dimensions with four different plains each pertaining to a single promoter and transcription factor will all intersect at a single point giving the specific concentration of each of the four ligands (and so on and so on).

Theoretical planned lab work send for synthesis the leader we have designed (in bio-brick format so that it would be modular and could be used with any combination of promoter and effector/reporter protein). Also send for synthesis the complimentary section of RNA design also in biobrick format for the same reasons.

Ligate the synthetic genes each to a well characterised promoter whose transcription is dependent on different ligands for instance PYEaR carg fusion (insert biobrick code name) and Pbad promoter (BBa_I0500). Ligate a fluorescent protein to the three prime end of the three loop system leader. Ligate the two fusions together to make a single insert transform E. coli with the plasmid. Grow up the cells in a range of concentrations of nitrate and a range of concentrations of arabinose as well as a number of different ratios. For instance colonies grown up in 0%, 0.1%, 0.2%, and 0.3% arabinose as well as 0mM KNO3, 5 mM KNO3, 10 mM KNO3 and 15 mM KNO3 as in the table below.

Substrate concentrations 0% 0.1% 0.2% 0.3% 0 mM KNO3 5 mM KNO3 10 mM KNO3 15 mM KNO3

WHAT IT IS (CONCEPT EXPLANATION), MATHS FOR IT, RUSSELL TO PRODUCE GRAPHICS, THEORETICAL LABS, GRAPH? Russell scenes as I'm on it I will put in much graphics I can and have a look at the theoretical labs