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Overview Sensitivity Tuner --- Characterisation --- Modelling Colour Generators --- Carotenoids (Orange/Red) --- Melanin (Brown) --- Violacein (Purple/Green) The Future Safety

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Week 6

Monday

Dry Work

Modelling

On friday, the two models for a proposed threshold switching system were created. Model 6.2 (competition between activator and repressor for a site on the DNA) was investigated; For different 'arabinose' (or other proposed input) and repressor concentrations the final level of output from the system was plotted. It was hoped that for different input levels (shown in the below graphs by the different green lines of output) the position of switching to low output would take place at different repressor levels due to the competitive nature of the system. Investigation into refining the model will take place. It is important to remember that whilst total output is what is seen, plotting rates of output production is necessary; the 'switching level' could be considered to be the point at which the rate of output production becomes zero. A standard way of designing this switching level is required.

Rate of output production against time for a series of set repressor levels showed that different steady rates of production were rapidly obtained (figure 1 below). The final rate of production (representative of the steady state) was plotted against repressor level, showing that the 'switching level' would occur in the region where the rate falls to zero (figure 2). Ideally, our system would have a well defined switching point, with a much sharper rise to high rates of output. This would likely be achieved by increasing hill coefficients, requiring a system with a greater degree of cooperativity.

Figure 1

figure 2

Ideally, for each set repressor level, the output will have a non-zero rate of production at different levels of arabinose input. The most useful plot here is rate of output against input concentration, with different set repressor levels as different lines on the same graph. This is equivalent to the competitive inhibtion of enzyme action, a sample plot of such a case is given below (figure 3).

figure 3

The feasibility of such a system investigated, the question is how to implement such a system?

To continue the modelling work; models need to be refined according to data that begins to come from the plate reader. A return to looking at the latch in terms of rates of output production will possibly be useful, although no such clean switching level is expected to be possible. Also, the preliminary plate reader data shows a decrease in rate of GFP production having reached a peak value. A possible explanation is the large requirements the high rates place on transcription and translational machinery of the cell. The overall effect could be considered to be one of negative autoregulation with potential for oscillations about a steady level, damping as the system progresses through time. Finally, this week we also aim to look at stochastic simulation possibilities using StochSim.

Wet Work

Amplification

After succesful PCR of pSB3K3, did a double digest (PstI and XbaI) of pSB3K3 (vector) and the following inserts: I746390, I746391, I746392, I746394, I746395. Ligated vector and each insert, transformed into Top10.

Melanin Retention

Inspection of the plates from Friday demonstrated no clear reduction of melanin efflux from the strains containing the multi-drug transporter knockouts (see below), thus it appears that the hypothesis that melanin efflux might be controlled by one of these two transporters doesn't stand up.

MG1655 containing an acrF deletion

MC1060 containing an acrAB deletion

Tuesday

Dry Work

Modelling

Thus far modelling has been rather preliminary. A systematic investigation of varying the parameters in a basic activator model is underway. There are nine parameters that can be changed relating to binding constants, hill coefficients and the rate of degradation of the activator protein.

Wet Work

Melanin pigment production

Examined the control plates made on Friday:

The picture clearly shows that the pigment production is due to the transformed cells, rather than conditions in the plate, or interactions between cell and the plate.

Amplification

Transformations of ligations were unsuccessful :( Positive ligation control was plated on the wrong antibiotic plate, so will repeat that tonight. Today de-bugged restriction digests. The gels showed about 50% ligation after 3 hours, so have repeated the double digest with another pSB3K3 sample and 380, 381, 382, 384, and 385, this time separating the DNA into 2 separate digests.

Another batch of overnight cultures of the 390, 391, 392, 394 and 395 constructs on the pSB1A2 plasmid were made up in preparation for another set of minipreps on the morrow, following exhaustion of the original miniprep stocks.