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Part:BBa_K081004:Experience
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Applications of BBa_K081004
Experiment 1 - performed by Lorenzo Pasotti and Mattia Quattrocelli
We performed this test assembly:
Description: we assembled K081014 (our RFP) under the Plux promoter that was contained into K081004. We kept pSB1AK3 as the scaffold vector.
Motivation: Plux promoter activity was studied in response to a constitutive expression of luxR. Plambda promoter without cI repressor guaranteed the constitutive expression of this gene. We expected to find a weak activity because luxR transcription factor is not active and so Plux cannot be turned on. We also expected to find a strong activity if we induce luxR activation using 3OC6-HSL.
Methods: we ligated K081014 downstream of K081004, transformed ligation into 50 ul of Invitrogen TOP10, plated transformed bacteria and screened 7 colonies to insulate a colony containing correctly ligated plasmids. We inoculated the positive colony into 9 ml of LB + Amp and incubated the culture at 37°C, 220 rpm for 15 hours. Then we diluted 1:10 the culture in two falcon tubes (5 ml cultures). One of these cultures was induced with 3OC6-HSL 1 uM. We let the two cultures grow for 2 hours and then we watched 50 ul at microscope through TRITC channel and DAPI channel for negative control.
Results: red fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture (see figure).
Comments: the pictures above were taken using the same gain and exposition time and with these parameters we cannot see any red fluorescent bacteria for the non induced culture, while we can see green fluorescent TOP10 in the induced culture. Increasing exposition time red fluorescence could be observed even for the non induced culture (last picture), confirming the weak activity of Plux promoter in response of unactive luxR. This experiment confirmed that Plux promoter has a weak activity without luxI (or 3OC6-HSL) and luxR protein is not sufficient to induce a strong transcription. Adding 3OC6-HSL, luxR becomes active and so Plux is turned on.
NOTE: K081004 has a point mutation in position 349 of C0062 coding sequence. This mutation changes the aminoacid, but luxR seems to work as expected. We also performed this test with an old version of K081004 carrying another point mutation (C->T at position 704 of C0062 coding sequence) that changed an aminoacid. Even in this case K081004 seemed to work as expected (results not shown).
Experiment 2 - performed by Lorenzo Pasotti and Mattia Quattrocelli
Description: this test is an extension of Experiment 1. We induced seven cultures with 3OC6-HSL at different concentrations to study quantitatively HSL-GFP static transfer function.
Motivation: we want to know cutoff point of this device.
Methods: the same as Experiment 1, but we diluted the overnight 9 ml culture 1:10 in seven falcon tubes (5 ml cultures). We induced the seven cultures with 3OC6-HSL 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 uM and 10 uM respectively. We incubated the cultures at 37°C, 220 rpm for 2 hours and then we watched 50 ul at microscope through TRITC channel and DAPI channel for negative control. We prepared two 50 ul glasses for each culture. We acquired three frames at 2.5 s (maximum exposition time) and 10 ms exposition time for every sample. We used ImageJ software to count automatically the number of cells for every acquisition. Then we computed n10/n2.5 (where n is the number of cells) to calculate the percentage of cells that glowed in the 10 ms acquisition, assuming that in the 2.5 s acquisition we can see the total number of cells. For each HSL concentration, we calculated the mean value of the 6 statistics (3 frames for each of the 2 glasses).
Results:
Comments: we computed the statistic n10/n2.5 because we know that when fluorescence is weak, cells cannot be seen at low exposition times. So, we calculate the ratio between the cells we can see at a low exposition time and the total number of cells in the frame. We chose 90 ms because the previous experiments with RFP had been performed using this parameter. We know that this is not an exact statistic, in fact we have to consider the count errors of ImageJ software, especially when cells are superimposed. Further quantitative experiments will have to be performed using standard measurement, in order to characterize parts.
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