Team:LCG-UNAM-Mexico/Experiments/Results

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Devices | Sensing Dispositive | Further Work  Devices Our system requires the constructions of two devices, the regulation device (BBa_K119010</a>) and the RcnA device (BBa_K119009</a>). We have finished the construction of the pBBR1MCS-5 plasmid that contains RcnA and also  we have cloned it into the YohM- mutant E.coli W3110 strain. Unfortunately we haven’t finished the construction of the regulation device because we have  had some problems with the ligation of the parts. This gel shows PCR products of RcnA(900bp) lanes 2-5. <img border="0" width="307" height="449" src="http://2008.igem.org/wiki/images/3/3f/260908.png" alt="260908.png"> This gel shows the double-digested pBBR1MCS-5 plasmid with EcoR1 -HindIII(Lane #1) and the Negative control with the pBBRMR1MCS-5 plasmid (Lane#2) <img border="0" width="189" height="349" src="http://2008.igem.org/wiki/images/c/c0/101008_rcnArest_plasmido_bueno.jpg" alt="101008_rcnArest_plasmido_bueno.jpg"> Once we had the YohM- strain transformed with the plasmid, we proceeded to tests the capabilities of measurement  of the instrument our colleague Martin Trejo built in order to measure the  resistivity in liquid media. <img src="http://2008.igem.org/wiki/images/c/cd/Boton_back.jpg" alt="Back to top" width="190" height="31" border="0"></a> <img src="http://2008.igem.org/wiki/images/9/99/Ribbon435773498.gif" border="0" width="579" height="9" /> </a> Sensing dispositive Once the electronic device was built, we investigated the range where it was able to measure nickel  concentrations on LB medium. We found that the device has maximum efficiency in the range (1e-7,5e-4). You can click on the image to see a larger resolution: <img src="http://2008.igem.org/wiki/images/b/bf/Ni_Res_loglogLR.jpg" width="560" height="350" border="0"></a> The graph shows that increasing the nickel level decreases resistivity which is in accordance with the ionic nature of nickel. Points represent the mean of three independent replicates and the line represents the linear regression model: <img align="left" src="http://2008.igem.org/wiki/images/a/a7/LR_Ni_re.png" width="230" height="30"> Where Ω is the resistivity measured in ohms and [Ni] is the nickel concentration.This shows that even in a complex medium (LB) we are able to detect changes in nickel concentration in a wide range that spans several order of magnitude. Then we investigated the time dynamics of resistivity in the presence of cells in the previously defined range of nickel concentrations. For this purpose we used cells lacking the rcnA gene, to reduce the effect of cells on our measurements. We have to take into account that even these cells add a variable to our measurements because they continuously import nickel to their cytoplasm. All our measurements lasted three minutes and we have one data point each 10ms, which means that we have 18000 data points for each experiment. The plot below shows means of each time point for two or three replicates at the indicated nickel concentration. The blue line represents experiments where nickel was not added. <img src="http://2008.igem.org/wiki/images/a/a9/NoNickelVSNickel.jpg" width="560" height="350" border="0"></a> The rapid increase that we observe in all the lines was observed in all our experiments independently of the conditions. We think that it represents a phase in which our electronic device adapts to the conditions; however, it is clear from the plot that the presence of cells does not disturb the change in resistivity that we observe We tried to estimate the rate at which nickel enters the cell by calculating the derivative of the previous curves on each point. The logic is that by substracting the derivatives of the blue curve to the other ones we would get the change in resistivity that is caused by nickel importing to the cytoplasm, i.e. the rate (flux) at which nickel enters the cells. However, we couldn’t find any significant relation despite reducing the interval to that with the most consistent replicates. In overall, we’ve shown that resistivity allow us to distinguish a wide range of nickel concentrations in a complex medium (LB and cells). More work is needed to standardize the protocols and to investigate the effect of other variables such as temperature. The fact that such good results were achieved with a home-made non-specific electronic device suggests that performance can be greatly enhanced by using specific electrodes. We also did some tests using the YohM-/pBBR1MCS-5/RcnA (BBa_K119009</a>) and the Wild Type W3110  E. Coli strain in LB with different concentrations of NiSO4. We observed that the replicates were slightly different between them and that some noise prevailed, even after the filters were applied. We think that we could improve this by making more replicates and by using specific electrodes. <img src="http://2008.igem.org/wiki/images/c/cd/Boton_back.jpg" alt="Back to top" width="190" height="31" border="0"></a> <img src="http://2008.igem.org/wiki/images/9/99/Ribbon435773498.gif" alt="ribbon" width="579" height="9" /> </a> Further Work As soon as we finish the construction of the regulation device we will make measures using the YohM-/pBBR1MCS-5/RcnA(BBa_K119009</a>)/Regulation  device(BBa_K119010</a>) in LB, LB with  NiSO4 and LB with NiSO4; using AHL. We will also try to make the measures in different liquid media because this maybe will help us expand our range of Nickel concentration detection by  measuring the resistivity. <img src="http://2008.igem.org/wiki/images/c/cd/Boton_back.jpg" alt="Back to top" width="190" height="31" border="0"></a> <img src="http://2008.igem.org/wiki/images/9/99/Ribbon435773498.gif" alt="ribbon" width="579" height="9" /> <img src="http://2008.igem.org/wiki/images/c/cd/Boton_back.jpg" border="0" width="190" height="31" /></a> <img src="http://2008.igem.org/wiki/images/4/44/Boton_exp2.jpg" border="0" width="190" height="31" /></a>  <img src="http://2008.igem.org/wiki/images/f/fc/Boton_exp3.jpg" border="0" width="190" height="31" /></a>  <img src="http://2008.igem.org/wiki/images/a/ad/Boton_exp4.jpg" border="0" width="190" height="31" />