Team:Imperial College London/Results

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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"|                  for catechol assays
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|valign="top"|'''Aims of experiment | '''In this experiment spectra from XylE expressing cells and XylE negative are compared after catechol addition to identify optimum absorption wavelength for quantification of output signal production  
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|valign="top"|'''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 spectra from the two cell cultures after catechol addition, reveals that in XylE expressing cultures a broad peak appears with maxima at 380nm  
'''Results | '''Overlay of the spectra from the two cell cultures after catechol addition, reveals that in XylE expressing cultures a broad peak appears with maxima at 380nm  
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Revision as of 00:29, 28 October 2010

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 spectra from the two cell cultures after catechol addition, reveals that in XylE expressing cultures a broad peak appears with maxima at 380nm

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

assay

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 2-step amplification performing better than 1-step amplifier. The prediction is that for our system 2-step amplification output will be visible around 4 minutes earlier than the1-step amplification system

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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 were acquired out of these assays of which the most important being the catechol(2,3)dioxygenase reaction profile with catechol. The graph generated delineate the course of the reaction in terms of yellow product production over time at various initial catechol concentrations

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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 the breakdown product to have a strong negative effect on cell survival.

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Experiment 5 | Characterizing GFP-XylE gene

product in whole cell cultures

Aims of experiment | In this experiment we wanted to test if our novel designed biopart, GFP- C(2,3)O fusion protein was functioning under the specification set by the team when designing this inactivated 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

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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 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

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Experiment 7 | Characterization in r.p.u. of

Pveg promoter

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).

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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 [[Team:Imperial_College_London/Modules/Detection_Module | 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.