Part:BBa_K081004:Experience

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Experiment 1 - performed by Lorenzo Pasotti and Mattia Quattrocelli

We performed this test assembly:

RFP protein generator under Plux

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).

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 was 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).

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