From 2012.igem.org

Welcome to the NRP UEA iGEM 2012 Wiki Projects Menu

Please choose the relevant link to view an overview of each project!

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.

Multiplication Circuit

Figure 1 Illustration of the effect of low tryptophan on a three loop system in the tryptophan operon (Madigan, et al., 2012)

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

There are four central mathematical operations: division, multiplication, addition and subtraction; the ability to do each in a cell unleashes massive potential for future applications. A multiplier effect can be produced by using a 'triple looped system' that naturally causes attenuation in the tryptophan operon. The tryptophan operon regulation works because it is found in bacterial systems, therefore meaning that transcription and translation occur simultaneously. The leader region (the section of RNA at the start of the mRNA that is not translated but has an effect on translation rate) contains 4 sites which have partial complementation. Region 1 has complementation to region 2, region 2 to 3 and region 3 to 4. When bonding occurs a central strand cannot bind to two strands simultaneously. This is seen in figure 1, where when binding between region 2 and 3 forms, a bond between region 3 and 4 does not form. Bond formation between region 3 and 4 causes transcription termination; it causes RNA polymerase to drop off. The hybridisation of region 2 and 3 prevents binding of 3 and 4. The reason for these different loops forming is due to the presence of rare tryptophan codons in region 1. In the absence of tryptophan, the tryptophan codons have nothing bound to them and the ribosome stalls at the site. Due to stalling of the ribosome, and continuation of transcription, a distance builds up which allows the formation of the 2:3 loop. In the presence of tryptophan, translation occurs until the stop codon is reached and only the 3:4 loop forms. 1:2 and 3:4 loops can form when no translation occurs (Platt, 1986). The reason for the 3:4 loop causing termination and the 2:3 loop not is that there is a difference in the stem and loop pattern. The removal of the ribosome binding site upstream means that the formation of the stem and loops are no longer dependent on the ribosome. Instead, the team designed a theoretical synthetic gene that would produce a mRNA strand that would complimentarily bind to site 1 causing the 2:3 loop to form and thus allowing transcription to occur to the termination codon. The probability of this happening is dependent on the concentration of the complimentary RNAs but also on the transcription initiation rate of the promoter. This creates a multiplier effect where the rate of transcription initiation in the promoter is multiplied by a number between 0 and 1.

Theoretical planned lab work

The team, should this idea have been taken further, would have sent for synthesis of the leader we have designed. And this synthetic gene would be ligated to well characterised promoters whose transcription is dependent on different ligands, for instance the hybrid (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.

table 2 data that would indicate the degree of knockdown

This would indicate the degree of transcriptional repression caused by the expression of the complimentary RNA it would also confirm the relationship between concentration of RNA complement, transcription initiation rate of promoter attached to the three loop system and and the rate of fall RNA transcript synthesis of the gene by observing harassments caused by fluorescent protein translated from RNA

Figure 3. Nth term equation for the proportion of transcription of the three loop system RNA that is attenuated by the expression of the complimentary RNA.

mathmatical modelling

This equation is in the nth term where each increment is increased conce Pn = proportion of transcripts that are bound by the complement RNA and attenuated at that level of transcription of the complementary Pn-1 = at one increment less transcription of R T = the binding half-life; the mean period for which the two RNAs are annealed. R = the rate of transcription initiation of the section of RNA complementary to sight 1 G = the volume of the cell S = the speed of RNA complement movement in the cell (temperature dependent)

Multi-Sensor System

Figure 4 Range of potential concentration ratios indicated by a particular transcription level (arbitrary) of two different promoters (1,2).

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.

Figure 5 each plane represents a range of possible concentration ratios between three ligands at a particular transcription rate (arbitrary). The intersection between the two different planes is shown as a purple line. The intersection line represents the range of possible ratios when only two planes are observed.

Figure 6 each plane represents a range of possible concentration ratios between three ligands at a particular transcription rate (arbitrary). The intersection between each pair both planes is indicated by a line. In the point where these lines intersect therefore is the point where all three planes intersect; this point gives the exact concentration of each of the three different substrates

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).

functional construction

there are a number of different ways of putting this theory into practice, either each of the different promoters selected can be fused to a fluorescent protein and cloned into different cells along with a constitutively expressed fluorescent protein (to control for metabolic and cell mass differences) and a homogenised sample split between each of the culture media containing fluorescent protein with a different promoters and the expression of each fluorescent protein measured using florimeter and mathematically analysed (see below) to give the exact concentrations of each of the ligands or all of the promoter - fluorescent protein fusions can be ligate it into one insert and transformed into a single cell (at this point the constitutively synthesised fluorescent protein is no longer necessary because comparison is made between expressions of different proteins within the same cell). This system has advantages and disadvantages. Because each cell has the full range of promoters necessary each cell can give a reading for the chemical concentrations in its direct vicinity meaning each cell can become a single “ pixel” which can make up a image of chemical concentrations throughout a sample. This would be useful in environments where diffusion rate is low and chemical concentrations vary e.g. soil. Exact concentrations could be calculated using laser microscopy but a simple photograph would yield much information about the system. Future lab work each promoter selected (in our case we have decided to work with small nitrogen species) must be characterised under different levels of and ratios of each of the ligands it will be measuring the concentration of. This data can then be used to analyse readings .

WHAT IT IS (CONCEPT EXPLANATION), MATHS FOR IT, RUSSELL TO PRODUCE GRAPHICS, THEORETICAL LABS, GRAPH? Russell seeings as I'm on it I will put in much graphics I can and have a look at the theoretical labs (Pascoe) ps there are two three-dimensional grafts to go in as well

Madigan, M T. Martinko, J M. Stahl, D A. clark (2012). Brock biology of microorganisms . 13th ed. London: Pearson. 232.