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
Currently there is limited data on Bs2 in the component library.In order to apply this reporter to practical use, we made contributions to supplement its characteristics, including excitation and emission wavelengths, unit fluorescence intensity in facultative anaerobes. In this study, we used the pET29a plasmid (J23119)(Fig.1)to express the Bs2 protein. In order to expand its application in facultative anaerobic bacteria, we used facultative anaerobic E.coli strain BL21 as expression vector to express Bs2 protein.(Fig.2) After 48 hours of cultivation, the excitation wavelength of Bs2 expressed by pET29a plasmid (J23119) was about 448nm and the emission wavelength was about 509nm(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
According to the measured excitation/emission wavelength, we measured the change of unit fluorescence intensity of E. coli BL21 introduced with pET29a-Bs2 plasmid (J23119) by controlling the culture temperature and time. After entering the logarithmic growth phase (OD600~0.5), IPTG was added to E. coli with pET29a-Bs2 plasmid (J23119) to induce Bs2 gene expression. OD600 and fluorescence intensity were measured every 1h, and OD600 and fluorescence intensity were measured every 2h after entering the stable phase.The unit fluorescence intensity of Bs2 in E. coli BL21 was determined (Fig.4).

Fig.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.

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]