Part:BBa_K5366073

J23119 promoter-RBS-Bs2-6xHis-T7 termonator

The Bs2 expression plasmid with J23119 as the promoter
(1) Excitation maximum and emission peak
To enhance the relative fluorescence unit intensity, we replaced the T7 promoter with the J23119 promoter. J23119 is a prokaryotic constitutive super strong promoter expression vector plasmid, which offers several advantages: Firstly, the J23119 promoter is one of the strongest constitutive promoters reported in its wild-type form, capable of efficiently driving the expression of foreign genes in E. coli. Secondly, the addition of an UP element (designated as UPa) upstream of the core J23119 promoter further enhances expression efficiency, resulting in a 1.34-fold increase in relative fluorescence units (RFU).
Currently, there is limited data available on Bs2 within the component library. To facilitate the practical application of this reporter, we aimed to enhance its characteristics, including excitation and emission wavelengths as well as unit fluorescence intensity in facultative anaerobes.
In this study, we utilized the pET29a plasmid (J23119) (Fig. 1) to express the Bs2 protein. To broaden its application in facultative anaerobic bacteria, we employed the facultative anaerobe E.coli strain BL21 as the expression vector for the Bs2 protein (Fig. 2). After 48 hours of cultivation, the excitation wavelength of the Bs2 protein expressed by the pET29a plasmid (J23119) was found to be approximately 448 nm, while the emission wavelength measured around 509 nm (Fig. 3).

Fig.1 Construction maps of plasmids of pET29a-Bs2(J23119)

Fig.2 Picture of solid mediumE. coli BL21 with pET29a-Bs2(J23119) on LB solid medium with kanamycin (10μg/ml) and IPTG (0.2 mM), incubated in 37℃ for 24h observed under UV and fluorescence microscopic image taken from the exponential growth phase when OD₆₀₀ was 0.8.

Fig.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.

(2)The expression of Bs2 protein in facultative anaerobic bacteria
Based on the measured excitation and emission wavelengths, we assessed the changes in unit fluorescence intensity of E. coli BL21 transformed with the pET29a-Bs2 plasmid (J23119) by controlling the culture temperature and duration. Once the culture reached the logarithmic growth phase (OD₆₀₀ ~ 0.5), IPTG was added to induce the expression of the Bs2 gene. Both OD₆₀₀ and fluorescence intensity were measured every hour during this phase. After entering the stable phase, these measurements were taken every two hours. The unit fluorescence intensity of Bs2 in E. coli BL21 was subsequently determined (Fig. 4).

Fig.4. RFU and RFU/OD₆₀₀ under the growth curve of E. coli with pET29a-Bs2 plasmid (J23119).The first is incubated at 20℃; The second is incubated at 30℃. The third is incubated at 37℃.Under incubation at 30℃ Celsius,RFU reached their maximum values.Under incubation at 30℃ Celsius,OD₆₀₀ reached their maximum values.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 287
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 520
  Illegal PstI site found at 287
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 287
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 287
  
• 1000
  COMPATIBLE WITH RFC[1000]