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Team:Imperial College London/Results
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|align="right"|[[Team:Imperial_College_London/Results/Exp6 | Learn More...]] | |align="right"|[[Team:Imperial_College_London/Results/Exp6 | Learn More...]] | ||
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| + | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"|Experiment 7 | Characterizing GFP-XylE gene | ||
| + | product in whole cell cultures | ||
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| + | |valign="top"|'''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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| + | |align="right"|[[Team:Imperial_College_London/Results/Exp7 | Learn More...]] | ||
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{| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | {| style="width:900px;background:#f5f5f5;text-align:justify;font-family: helvetica, arial, sans-serif;color:#555555;margin-top:5px;" cellspacing="20" | ||
|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"| Testing the Detection Module | |style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"| Testing the Detection Module | ||
Revision as of 22:50, 27 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 cells and XylE negative are compared after catechol addition 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 | |
| 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 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 | |
| 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 the breakdown product 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 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 | |
| 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 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 | 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 | |
| Learn More... |
| Testing the Detection Module |
| We had huge problems cloning the cell surface protein construct into B. subtilis. 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. |
| 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 [http://ukpmc.ac.uk/backend/ptpmcrender.cgi?accid=PMC40587&blobtype=pdf 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. |
