Building on BBa_K4119002, we modified the promoters and plasmid to express the protein Bs2, a fluorescent protein reliant on flavin mononucleotide (FMN). Unlike green fluorescent protein (GFP), which necessitates oxygen for fluorescence emission, Bs2 is an oxygen-independent fluorescent protein that can function in an anaerobic fluorescence gene reporting system [1]. To optimize the expression of Bs2, we replaced the J23119 promoter and assessed its characteristics, including excitation and emission wavelengths, unit fluorescence intensity in facultative anaerobes, and imaging of facultative anaerobes harboring recombinant plasmids under UV light.
In this study, we utilized the recombinant plasmid pET29a-Bs2 (J23119) to express the Bs2 protein (Fig 1). For broader applications in facultative anaerobes, we employed the facultative anaerobic
Figure 1 Construction maps of plasmids of pET29a-Bs2(J23119)
Figure 2 E.coli BL21 with pET29a-Bs2 (J23119) on LB solid medium with kanamycin(10μg/ml) and IPTG (0.2 mM),incubated in 37 ℃ for 24h. All was observed under UV
Figure 3 Excitation maximum and emission peak (RFU: Relative fluorescence unit)
The Ex Wavelength in nm (Em: 520) indicates that there is one peak values of excite wavelength and it is 448 nm. The Em Wavelength in nm (Ex: 448 nm) shows excluding the impact of three peaks value of excite wavelength, the emission wavelength is around 509 nm.
Finally, based on the measured excitation and emission wavelengths, we adjusted cultivation temperature and time to evaluate changes in unit fluorescence intensity of E. coli BL21 with the pET29a-Bs2 (J23119) plasmid. Upon reaching the logarithmic growth phase (OD600 =0.5), IPTG was added to induce Bs2 gene expression. OD600 and fluorescence intensity were recorded hourly until steady state, after which measurements were taken every two hours. The unit fluorescence intensity of Bs2 in E. coli BL21 with recombinant plasmids pET29a-Bs2 (J23119) was determined (Fig. 4)
Figure 4 RFU and RFU/OD600 under the growth curve of E. coli with pET29a-Bs2 plasmid (J23119)
(A) is incubated at 20 ℃; (B) is incubated at 30℃. (C) is incubated at 37 ℃. (A)-(C) indicate that OD600 has the minimal fluctuation at 30 ℃, however at 30 ℃ RFU maintained the highest than the other two.
1. Chi Cheng, Meng Lin, Wenyan Jiang, Jingbo Zhao, Weiming Lib,, Shang-Tian Yang (2019) ‘Development of an in vivo fluorescence based gene expression reporter system for Clostridium tyrobutyricum.’, Journal of Biotechnology, 305, pp18-22.