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Part:BBa_K081022:Experience
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Applications of BBa_K081022
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.
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. 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.
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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UNIPV-Pavia iGEM 2011 |
K081022 has been characterized through the measurement systems:
The inducible device BBa_K081022 was assembled upstream of different mRFP coding sequences, containing an RBS from the Community collection. The assembled RBSs are:
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range. The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (Scell) and R.P.U.s (Relative Promoter Units) as explained in measurements section.
The Hill function relating the induction to the Scell is reported below: Scell=α * ( δ + (1-δ)/(1+(K/Induction)η) ) Operative parameters of the promoter are derived from the estimated Hill equations obtained by nonlinear least squares fitting (lsqnonlin Matlab routine) of the Hill function expressed in RPUs:
The estimated parameters for the Hill functions are summarized in the table below. For more details on parameter estimation, see the model section.
Data are provided as average [CV%]. From this table, it is evident that, whilst αpLux assumes significantly different values for different RBSs, ηpLux and kpLux assume very similar values. This result shows that RBS variation modulates the amplitude of Hill function, not affecting the shape of the curve. The four induction curves result to be the same Hill function modulated in amplitude by a parameter, that is the estimated RBS efficiency for this measurement system. These results are quite encouraging, because suggest that, given the non-modular behavior of RBS dpending on the encoded gene, the RBS has a modular behaviour respect to the promoter. The operative parameters are summarized in the table below:
These operative parameters provide useful information on the behavior of this 3OC6-HSL inducible device. RPUmax assumes very different values in terms of RPUs. This can't be explained by RBS modulation, since RPUs have been evaluated by normalizing Scell of pLux-RBSx for the one of J23101-RBSx. It is evident that some nonlinear effect on maximum strength, maybe due to saturation phenomena on protein expression, occur. The same RPUs should be observed for every RBS, since the normalization by the standard reference used for RPUs computation should eliminate the RBS contribute. Here different RPUs are observed, maybe due to nonlinear RBS behavior or to saturation phenomena occurring with this very strong promoter. The switch point and linear boundaries are quite constant in all the cases, showing that the linear region of this system is not affected by RBS changes.
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