Team:XJTU-CHINA/Measurement
Measurement
Our project employed pJ23106 as a constitutive promoter of a photosensitive protein -- Cph8 (BBa_K2598006) which is the key part of our light-control system, so we plan to quantitatively characterize this promoter BBa_J23106 in order to acquire parameters in realistic conditions which can be used in modelling. We used part BBa_K1819006, which is consist of GFP (BBa_K1819000) and its promoter J23106. We have experimentally validated this part in three different chassis of Escherichia coli: DH5α, S17-7 and BL21, which are quite commonly used in the lab.
Meanwhile, we also conducted the scanning of excitation wavelength and emission wavelength of this GFP at different of time point, trying to obtain the precise emission and excitation spectra in the conditions of our equipment, and evaluate the possible influence of hosts on its spectra. This validation will facilitate the measurements of this GFP with different regulatory parts we assembled.
Furthermore, in order to make our results more reliable and comparable, we prepared the standard curves of OD600 and fluorescein based on the iGEM Kit and protocols, which will benefit the precise and quantitative measurement of these two parameters in synthetic biology.
The protocol for bacteria cultivation and fluorescent intensity test parameters as well as standard curve protocols are listed below, and Figure 1 ~Figure 6 were achieved.
- Day1: Transform pSB1K3-K1819006 into Escherichia coli DH5α, S17-7 and BL21 respectively.
- Day2: Inoculate the transformants in 5-10 mL LB medium with chloramphenicol (25 ug/ml) which was cultured for 12h.
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Day3:
1. Inoculate 1% of the recombinant of E.coli into fresh LB medium with chloramphenicol, and culture at 37℃,200 rpm
2. Measure OD600 and GFP fluorescent intensity (RFU) of the bacteria every 2 hours using 200 ul of the culture broth with six parallel groups.
3. We will test (1) OD600, (2) GFP emission scan curve from 495 nm to 580 nm when the excitation wavelength was fixed at 475 nm, (3) GFP excitation scan curve from 430 nm to 520 nm with the fixed emission at 545 nm.
4. For preparation of standard curve of OD600 and fluorescein:
The results are as follows:
As shown in Fig 1, all the three E. coli experienced the basic cell growth of very short lag phase, fast log phase and quite long stationary phase during 60-h cultivation. OD600 of all three strains basically reached stability after about 14h cultivation, and it is clearly showed that BL21 had the most vigorous growth, S17-1 the second, while DH5α had the slowest growth, but there was no significant difference between these three, in case of lab-scale cultivation.
The GFP intensity changes per cell were also be compared between these three strains, which shared the similar trends with cell growth. The GFP intensity per cell turned to be stable after about 12h cultivation. And recombinant S17-1 exhibited highest intensity among three strains, while BL21 is the least, indicating S17-1 somehow could be a better host for the measurement of this GFP-harboring parts based on our results.
From Fig 1 it is clear to show that the intensity of GFP was gradually increased during the 60h cultivation in all three strains, and the wavelength with highest emission located around 515 nm when the excitation wavelength was fixed at 475 nm. There was no significant difference in the spectra profile of GFP between three E. coli strains, while the intensity of GFP varied in three strains, with the highest intensity in S17-1 at 3.9x107, and lowest in BL21 at 2.0x107.
When the scanning of excitation wavelength was performed with emission wavelength was fixed at 545nm, similar results were achieved. The intensity of GFP was stably increased and the wavelength with highest excitation was around 505 nm when the emission wavelength was fixed in all three stains. And the three strains shared quite similar spectra profile of GFP, but the intensity of GFP differed, with the highest intensity in S17-1 at 2.6x107, and lowest in BL21 at 1.3x107.
All in all, our validation of the emission and excitation spectra of this GFP in three different recombinant E.coli, provides us the precise wavelength for testing of our parts, as well as demonstrated that the host will not affect the spectra profile of GFP but influence the intensity.
As shown in Fig 5 and Fig 6, the standard curves exhibited good linear relation with the concentrations. These standard curves will be helpful to convert arbitrary units to calibrated standard units.