Part:BBa_K081022:Experience

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Applications of BBa_K081022

GFP protein generator under Plux and constitutive expression of luxR

Description: we assembled K081012 (our GFP) under the Plux promoter that was contained into K081022. We kept pSB1A2 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 K081012 downstream of K081022, transformed ligation, plated transformed bacteria and screened three 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 FITC channel and DAPI channel for negative control.
Results: green fluorescent cells could not be observed at microscope (see figure) for the non induced culture, while they could be observed for the induced culture.

Non induced culture: TOP10 cells cannot be seen at microscope

Induced culture: fluorescent TOP10 cells at microscope

Same frames as above, but superexposed: non induced (left) and induced (right) cultures

Comments: the pictures above were taken using the same gain and exposition time as the previous experiments and with these parameters we cannot see any green fluorescent bacteria for the non induced culture, while we can see green fluorescent TOP10 in the induced culture. As we wrote for TEST 6 and TEST 7, increasing exposition time green 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.

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