Part:BBa_K3580003:Experience
Applications of BBa_K3580003
Waseda 2020
In our project, we inserted this parts into pSB1C3 plasmid.
In order to compare these parts(BBa_K3580003 and BBa_K934025), we first measured the fluorescence of GFP expressed in vivo. Each part was transformed into an E. coli JM2.300 strain with luxR plasmid introduced into that. After overnight incubation, diluted and OD600-matched to fresh Culture and 3OC6HSL induction was added to the corresponding cultures of both parts at the appropriate OD600. After that, the OD600 of the culture was measured every 1h and the GFP fluorescence values in vivo were measured by diluting aliquot for measurement from the culture to a constant turbidity (OD600=0.4) at each measurement (Fig1).In the sample name of Fig1, the mark (+) means adding 3OCHSL and (-) means not. The measured GFP fluorescence values of the final time point (240 min) from the start of induction at each condition are shown in Fig 2. The fluorescence of tagged GFP (BBa_K3580003) was lower than that of normal GFP(BBa_K934025) at 240 min point (Fig1). Although GFP is a stable protein with a β-barrel and much difficult to be degraded, this result shows that tagged GFP was successfully degraded in vivo as we desired.
Then, we compared the fluorescence of GFP in a cell-free system which was extracted from the E.coli containing luxR protein (Fig 3). Because of the programmed degradation, the fluorescence of tagged GFP(BBa_K3580003) showed slight signal nearly equal to a negative control where neither the template GFP DNA nor the inducer AHL exists. Those results show that ssrA tagged protein can be degraded much both in vivo and in vitro. Based on this data, we modified the degradation terms in the model, as described below.
Material and Method: Waseda 2020
In vivo measurement O/N->Fresh Culture->Induction Each part was transformed into an E. coli JM2.300 strain with luxR plasmid introduced into that. We prepared overnight culuture of each cell (plux/tet-GFP, plux/tet-GFP-LVA, plasmid nega) in LB 3ml scale with each antibiotics(plux/tet-GFP and plux/tet-GFP-LVA: Cm 25 µg/ml+Kan 30 µg/ml, plasmid nega: Kan 30 µg/ml) at 37℃ for 12h. After overnight incubation, diluted and OD600-matched to fresh Culture. We took 30 µl(from plux/tet-GFP, plux/tet-GFP-LVA, plasmid nega) of the overnight culture of inducer cell into fresh 3ml LB with each antibiotics and cultured until the observed OD600 reaches around 0.50. 3OC6HSL induction was added to the corresponding cultures of both parts at the appropriate OD600.
Measurement
After induction, the OD600 of the culture was measured every 1h and the GFP fluorescence values in vivo were measured by diluting aliquot for measurement from the culture to a constant turbidity (OD600=0.4) at each measurement.
We diluted all aliquot for measurement with LB to OD600=0.4. All samples were diluted to 100 µl. The total volume of 100 μl was dispensed into 96-well plates (Iwaki) and green fluorescence values were measured (excitation wavelength: 473 nm, emission wavelength: 535 nm) using FLA5100 (Fujifilm).
We performed the above experiment in the same way from three different colonies transformed on LB agar medium, with N=3 for the number of samples.
In vitro measurement
1.Prepare the cell extract for Cell-free system containing reporter protein.We prepared cell extracts containing the luxR reporter protein according
2.LVA degradation tag assay
Under each condition, the reaction composition solution was prepared as shown in Table 1. Cell-free reactions were performed on a 20µl scale. The fluorescence of cell-free expressed reporter GFP was measured on a real-time PCR(Step One Plus Real-Time PCR System, Applied Biosystems, Mx3005P, Stratagene California) for 12 hours at 37°C and the measured fluorescence values were quantitatively calibrated by FITC.
MixA is a mixture of small molecules mainly required for translation and its composition is shown in Table.2 below.
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