Team:Imperial College London/Results

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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;"|Results
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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"
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"|Experiment 1 | Optimum absorption wavelength
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<img style="width:250px;border:solid 5px #555555" src="https://static.igem.org/mediawiki/2010/e/e6/Spectra_of_Xyle_cells.jpg" />
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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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|-
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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.
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'''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.
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|align="right"|[[Team:Imperial_College_London/Results/Exp1 | Learn More...]]
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__NOTOC__
 
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=Cat-O2-lase Protein=
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The XylE gene, originating from the TOL plasmid of Pseudomonas putida, was the choice for the output signal mediator of our system. The system is based on catechol 2,3-dioxygenase enzyme. Colonies of cells that express xylE gene product become yellow within seconds after selection plates are sprayed with catechol, a colorless substrate, that is converted by CatO2ase to the yellow product, 2-hydroxymuconic semialdehyde. This reporter system is ideal for our project as it has very fast kinetics, gives a visual output by naked eye and the substrate used is very cheap.
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"|Experiment 2 | The threshold value of catechol  
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assay
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<img style="width:250px;border:solid 5px #555555" src="https://static.igem.org/mediawiki/2010/3/38/CS1.JPG" />
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</html>
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|-
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|valign="top"|'''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.
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'''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.
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|-
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|align="right"|[[Team:Imperial_College_London/Results/Exp2 | Learn More...]]
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|}
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==Characterization of CatO2lase protein==
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"|Experiment 3 | Characterizing kinetic parameters
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of C(2,3)O in whole cells
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<img style="width:250px;border:solid 5px #555555" src="https://static.igem.org/mediawiki/2010/c/cb/IC_Assay_3_sept.jpg" />
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</html>
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|-
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|valign="top"|'''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.
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'''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.
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|-
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|align="right"|[[Team:Imperial_College_London/Results/Exp3 | Learn More...]]
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|}
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==Experiment 1 - Determining optimum absorption wavelength for catechol assays==
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The CatO2lase protein, except as an ideal output signal for our engineered bacterial detector it can also serve as a great reporter gene. Its characteristics can be exploited in a wide range of fields across biological sciences field, so it was one of the team's first candidates for further characterization. Qualitative and quantitative data were gathered and presented. All further tests involving XylE transformed cells were quantified by measuring absorbonce at 380nm wavelenth.
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"|Experiment 4 | Assaying cell-growth in presence
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of Catechol
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<img style="width:250px;border:solid 5px #555555" src="https://static.igem.org/mediawiki/2010/d/dd/XylE_M9_Growth_%28600%29.jpg" />
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</html>
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|-
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|valign="top"|'''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.
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'''Results | ''' Experiments indicate that the breakdown products to have a strong negative effect on cell survival.
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|-
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|align="right"|[[Team:Imperial_College_London/Results/Exp4 | Learn More...]]
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|}
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[[Image:CS1.JPG|thumb|right|200px]]
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Catechol ,the initial substrate of CatO2las enzyme, is colourless. However within seconds of its addition the colonies/liquid cultures of XylE expressing cells become yellow, indicating production of product which absorbs light in the region visible spectrum. An spectrophotometric assay was prepared, where the spectra of two cultures of E.coli (one XylE gene transformed and the other not)were compared on addition of 0.1mM Catechol substrate. The spectra (figure 1) showed that in XylE transformed cells, a broad peak appears at about '''380nm'''. The absorbance of the particular wavelengths of light by the product of the enzymatic reaction 2-hydroxymuconic semialdehyde, is what causes the yellow output.
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"|Experiment 5 | Characterizing GFP-XylE gene
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[[Image:Spectra of Xyle cells.jpg|thumb|center|500px|Figure 1: Spectra of cultures of cells -ve and +ve on XylE gene. Note the broad peak in the spectra of Xyle transformed cells, which is centered around 380nm.]]
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product in whole cell cultures
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|style="width:250px;" rowspan="2" align="center"|<html>
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<img style="width:250px;border:solid 5px #555555" src="https://static.igem.org/mediawiki/2010/0/0d/GFPXylE.jpg" />
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</html>
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|-
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|valign="top"|'''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.
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'''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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|-
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|align="right"|[[Team:Imperial_College_London/Results/Exp5 | Learn More...]]
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|}
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References
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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"
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*Chromogenic identification of genetic regulatory signals in Bacillus subtilis based on expression of a cloned Pseudomonas gene. Zukowski, M.M., Gaffney, D.F., Speck, D., Kauffmann, M., Findeli, A., Wisecup, A., Lecocq, J.P. Proc. Natl. Acad. Sci. U.S.A. (1983)
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"|Experiment 6 | In vitro characterization of C(2,3)O
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in cell lysate
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|style="width:250px;" rowspan="2" align="center"|<html>
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<img style="width:250px;border:solid 5px #555555" src="https://static.igem.org/mediawiki/2010/e/eb/Grapfit_curve_1.jpg" />
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</html>
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|-
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|valign="top"|'''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
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'''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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|-
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|align="right"|[[Team:Imperial_College_London/Results/Exp6 | Learn More...]]
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|}
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==Experiment 2 - The threshold value of catechol assay==
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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"
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[[Image:PA190014.JPG|thumb|right|200px|Threshold value experiment was conducted on a 96 well plates. Yellow wells are the positive tests, LB color and M9 color mediums wells are the negative tests. ]]
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"| Experiment 7 | Characterization  of Pveg promoter
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Addition of catechol to a a culture of cells constitutively expressing C(2,3)0 enzyme results  into production of yellow colonies on plate or yellow liquid cultures if E.coli is grown in liquid medium. In a lab where a spectrophotometer is readily available anyone can accurately quantify production of even the smallest quantity of the yellow 2-hydroxymuconic semialdehyde product. However, as our biological detector is intended for field use where sophisticated spectroscopic equipment is not available, it was obvious that we needed to establish the threshold value at which a positive result (yellow color) would be detectable by the naked eye.
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in r.p.u.
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<img style="width:250px;border:solid 5px #555555" src="https://static.igem.org/mediawiki/2010/f/ff/Untitled_5.jpg" />
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</html>
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|-
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|valign="top"|'''Aims of experiment | ''' Measuring the activity of BioBrick promoters using an in vivo reference standard.  
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'''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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|-
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|align="right"|[[Team:Imperial_College_London/Results/Exp7 | Learn More...]]
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|}
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The set up of this experiment involved preparation of XylE expressing E.coli cultures at an O.D. of 0.5 in LB and M9 medium. Then a gradient of catechol concentrations was prepared and added to the cell cultures. A group of 5 individuals, but not the lab member preparing the test samples, were given the test cultures and asked where on the gradient they were not able anymore to see the yellow color. The 2 concentrations where the transition from yellow to colorless occurred were identified and a second gradient of catechol concentrations was prepared. In this second gradient the gap between the 2 concentration was analysed. The highest catechol concentration was the least yellow from the first part of the experiment, while the lowest catechol concentration was the first "colourless" concentration from the first part of the experiment. Again the group of 5 testers identified the transition concentrations from yellow to colorless.
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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"
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"| Testing the Detection Module
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The threshold concentration of catechol needed for a person to identify a yellow positive test was found to be '''30-40μM'''.
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|-
 +
|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 [[Team:Imperial_College_London/Software_Tool | Software Tool]]!
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|}
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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"
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|style="font-family: helvetica, arial, sans-serif;font-size:2em;color:#ea8828;" align="left"| Testing the Signaling Module
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|-
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|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.  
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==Experiment 3 - Characterising kinetic parameters of C(2,3)O in whole cells ==
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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.
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[[Image:Xyle reaction pic.jpg|thumb|right|300px|Catechol converted to 2-hydroxymuconic semialdehyde by C(2,3)lase]]
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|}
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In our Parasight project the XylE gene product, C(2,3)lase enzyme, is the means by which an output signal is generated. If the parasite is present is the water supply, the bacterial system is activated by an input signal which produces the yellow color, the positive output signal. Test assays in the lab on colonies of cells expressing XylE gene, become yellow only seconds after addition of catechol (100mM). From this it was realized our group that the kinetics of the C(2,3)lase enzyme are such that make it an attractive candidate  as a reporter enzyme. So, we decided that further characterization of the kinetics of the C(2,3)lase might help identify a really efficient reporter for research centers.
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[[Image:Catechol assay graph (0.2-1).jpg|thumb|right|300px|The first catechol assay data analysis. The assay involved addition of catechol concentrations of 0.2, 0.4, 0,6 0.8 and 1mM to 0.5 O.D. of XylE transformed E.coli cell cultures. The XylE gene was under the influence of J23101 promoter.]]
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In this experiment we investigated how the velocity of the reaction (catechol converted to 2-hydroxymuconic semialdehyde by C(2,3)lase) varies with increasing initial concentrations of catechol, the substrate. Overnight cultures of XylE transformed E.coli were plated on a 96 wells plate at an optical density (O.D.) of 0.5 units of absorbance. (The optimum initial O.D. of cells was determined in a previous experiment. This O.D. may vary across different labs as the sensitivity of the spectrophotometer/plate reader is a factor). Catechol concentrations ranging from 0.1-1mM were made from 100mM stock solution and added to the cells of the 96-well plate. The velocity of the reaction was monitored using the plate reader spectrophotometer as the reaction is directly proportional to the production of yellow color. The yellow output was measured at 380nm wavelength, while cell density at 600nm. The XylE gene expression was under the influence of J23101 promoter.
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[[Image:IC_Assay_3_sept.jpg|thumb|right|300px|Catechol assay on XylE-trasformed cells in a 96-well plate (A to H decreasing cell concentration, 1-10 decreasing catechol concentration, column 11 and 12 negative and control)]]
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The results of the experiment confirmed previous hints of the huge catalytic turnover rate of the C(2,3)0 enzyme. The graph of the right illustrates production of yellow product per min. The slope at the initial stages of the reaction (first minutes) would represent the rate of the reaction, that is the initial Velocity. By calculating initial velocities of the reaction at different substrate concentrations, one can plot a Michaelis-Menten curve to extract kinetic parameters such as Vmax, Km and catalytic turnover number.
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Several attempts were made to determine these parameters on whole cells. Unfortunately due to the actual nature of the enzyme, velocity of reaction and technical limitations of lab equipement this has been proven impossible. To start with one of the limitation was that the color output and the extinction coefficient of the yellow product is so large, that the system saturates within the first minutes after catechol addition to cell cultures. The spectrophotometer and plate reader reaches its detection maximum reading value from the start of the reaction. Also due to the high velocity of the reaction, in the time frame of about 10 seconds that are needed to load the plate into the plate reader after catechol addition, the reaction has already proceeded too far. So initial velocity measurements of the reactions needed for construction of Michaelis-Menten curve are inaccurate. These problem could be overcome by decreasing total enzyme concentration. In whole cell cultures this would be possible by decreasing cell density in each well as each cell contain an average C(2,3)O enzyme concentration. However, previous experiments showed that cell cultures with optical densities lower than 0.5 O.D. introduce serious perturbations in the readings of the spectrophotometer and plate reader detectors.
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Despite these limitations some useful data were extracted out of these assays. More specifically we acquired data that delineate the course of the reaction in terms of yellow product production over time at various catechol concentrations. The results of one of these assays is presented in the figure below. In order to extract data that will allow characterization of the kinetic parameters of catechol dioxygenase enzyme our lab team proposes purification of the protein from cell lysate and in vitro characterization.
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[[Image:HMS prod. over time curve for xylE.jpg|thumb|600px|center|Graph shows production of HMS (yellow product) over time after catechol addition at time 0 minutes. Different curves represent different catechol concentration added to the cell cultures.]]
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==Experiment 4==
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[[Image:IC Module3.JPG|thumb|right|400px|The GFP-XylE gene construct]]
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In paper blabla.... In our construct, GFP was added to the N-terminal of C(2,3) monomer. C(2,3) enzyme is a homotetramer were all 4 subunits are required for its activity. We hoped that by adding GFP at the N-terminus of the catechol dioxygenase enzyme, we would block tetramerization of the enzyme, thus render it inactive. This modification is important as it makes our system inducible. The cleavage of the GFP by TEV protease, on arrival of input signal, means that Catechol dioxygenase becomes active only when the parasite is detected.
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-
 
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To investigate this, an experiment was designed where catechol was added to XylE transformed E.coli. The velocity of the reaction was monitored at various initial catechol concentrations. The experimental set up is essentially the same as described above for determining the kinetics of C(2,3)O enzyme. The only difference is that E.coli cells used are the ones transformed with GFP-XylE gene construct instead of XylE gene.
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Latest revision as of 02:43, 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 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

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 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.

Retrieved from "http://2010.igem.org/Team:Imperial_College_London/Results"