Team:Imperial College London/Results


Experimental Results Exp 1 | Exp 2 | Exp 3 | Exp 4 | Exp 5 | Exp 6 | Exp 7
Testing is a fundamental stage of the engineering design cycle and is a crucial part of charactrising BioBrick Standard Biological Parts so that other people can benefit from our work. We've compiled all our results on this page, detailing how the experiments were carried out and the significance of the data.
Experiment 1 | Optimum absorption wavelength
for catechol assays
Aims of experiment | In this experiment spectra from XylE expressing and XylE negative cells are compared after catechol addition. This allowed us to identify optimum absorption wavelength for quantification of output signal production.

Results | Overlay of the two spectra from the cell cultures after catechol addition, reveals a broad peak appears with maximum at 380nm unique to XylE expressing cultures.

Learn More...
Experiment 2 | The threshold value of catechol


Aims of experiment | In this experiment we wanted to establish the threshold value at which a positive result (yellow color) would be detectable by the naked eye.

Results | The threshold concentration of catechol needed for a person to identify a yellow positive test was found to be 30-40μM. This result was fed back to the modelling team and allowed them to constrain the concentration values of catechol to be used. Furthermore, it facilitated the determination of conditions for modelling of 2-step vs 1-step amplification. Two step was shown to be better. The prediction for our system is that 2-step amplification output will be visible around 4 minutes earlier than the 1-step amplification system.

Learn More...
Experiment 3 | Characterizing kinetic parameters

of C(2,3)O in whole cells

Aims of experiment | In this experiment we attempt to characterize the kinetic parameters of catechol(2,3) dioxygenase enzyme, an existing registry part in whole cell cultures.

Results | Very useful information was acquired out of these assays of which the most important is the reaction profile of catechol(2,3)dioxygenase with catechol. The course of the reaction was analysed indirectly from the yellow product production over time at various initial catechol concentrations.

Learn More...
Experiment 4 | Assaying cell-growth in presence

of Catechol

Aims of experiment | In order to assess possible effects of either Catechol or the breakdown product 2-hydroxymuconic semialdehyde, we performed growth assays under a variety of conditions.

Results | Experiments indicate that the breakdown products to have a strong negative effect on cell survival.

Learn More...
Experiment 5 | Characterizing GFP-XylE gene

product in whole cell cultures

Aims of experiment | In this experiment we wanted to test if our custom designed biopart, GFP - XylE fusion protein, functions as expected. The specifications were set by the team to design an inactivated XylE reporter enzyme.

Results | Comparing reaction profile graphs of cell cultures of XylE expressing cells Vs GFP-XylE expressing cells showed that the designed reporter enzyme has more than 10 fold decreased activity in comparison to wild type reporter enzyme catechol(2,3)dioxygenase.

Learn More...
Experiment 6 | In vitro characterization of C(2,3)O

in cell lysate

Aims of experiment | In this experiment we attempt to characterize in vitro the kinetic parameters of the enzyme catechol(2,3)dioxygenase (XylE) in cell lysate

Results | Construnction of Michaelis-Menten curve and determination of kinetic parameters of catechol(2,3)dioxygenase enzyme in E.coli. The calculated Km value - the substrate concentration at which velocity is half of the maximum, is 0.71mM of catechol. The Vmax is 3.37mM/min at x20fold dilution of cell lysate.

Learn More...
Experiment 7 | Characterization of Pveg promoter

in r.p.u.

Aims of experiment | Measuring the activity of BioBrick promoters using an in vivo reference standard.

Results | pVeg promoter in pSB1C3 vector, a high copy plasmid, has an 1.62 r.p.u value and in 3K3 vector, a low copy plasmid an 0.79 r.p.u. value. These values were derived by dividing signal from the production of HMS by the pVeg promoter population of cells by signal from the standard promoter J23101 (r.p.u value of 1).

Learn More...
Testing the Detection Module
Cloning the cell surface protein construct into B. subtilis has been problematic, which means we have not had time to upload testing results yet. However, we hope to have done this by the Jamboree. By increasing NaCl concentration to 1M, the electrostatic interactions will be disrupted and we can use a nickel column to bind the His-tagged surface protein thereby ensuring that it correctly attached to the cell wall.

We then hope to test each cell surface linker by adding TEV protease and measuring how much AIP is cleaved. The linker that gives us the highest degree of cleavage will be used for the final surface protein for cleavage by the Schistosoma elastase. The characterisation of the linkers in the surface protein are definitely an area for future work, as the surface protein has a vast number of potential applications. For more information, check out our new Software Tool!

Testing the Signaling Module
Due to problems with the synthesis of this construct, we only received it at the end of Week 16 and therefore have not had sufficient time to clone it and test it. However, this module would have been tested with a synthetic AIP, which has successfully been used in previous experiments, such as by An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae by Havarstein, Coomaraswamy & Morrison. This would have activated the signaling cascade, resulting in the phosphorylation of ComE, which would then be able to induce transcription of a reporter gene.

However, this step would have been relatively straightforward and we have no reason to believe it would not function properly in B. subtilis, as like the organism from which the signaling system is taken (S. pneumoniae), it is Gram positive and has been shown to signal via AIPs in quorum sensing systems.