Part:BBa_K4119002

flavin mononucleotide (FMN)-dependent fluorescent protein Bs2

Bs2 is one of the flavin mononucleotide (FMN)-based fluorescent proteins. We use 450nm as excited wavelength and 500nm as absorption of emission wavelength.
To find more about this flavin mononucleotide (FMN)-based fluorescent protein, view doi:/10.1016/j.jbiotec.2019.08.019

Results

In our project, the key promoter vgb was a microaerobic induced promoter of Vitreoscilla hemoglobin gene. Considering the gene compatibility difference between different host bacteria, we designed the pMTL-Pvgb-bs2 plasmid to determine whether the promoter vgb could work normally in Clostridium tyrobutyricum by detecting the fluorescent expression intensity of fluorescent protein Bs2.
The green fluorescent protein (GFP) has been one of the most widely used reporter in bioprocess monitoring of gene expression. However, they are not functional under anaerobic conditions, and thus cannot be employed as reporters in Clostridium. A series of flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) have been reported, which could exhibit strong signals in the absence of O₂. FbFPs have been successfully used as a fluorescent label in anaerobic or facultative anaerobic bacteria, including several species of Clostridium for monitoring of protein expression, evaluation of promoter strength, and for proof-of-concept demonstration of transcriptional repression, etc.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 215
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 215
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 215
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 215
  
• 1000
  COMPATIBLE WITH RFC[1000]

Group: Nanjing-BioX

Author: Yuyao Cao, Yijiu Lu

Summary: Characterization of the transcription of Bs2 gene regulated by Pfba promoter

<Characterization from Nanjing-BioX:

We constructed pMTL-Pfba-Bs2 plasmid using Pfba promoter and Bs2 gene, transformed the plasmid into Clostridium tyrobutyricum (C. tyrobutyricum) and detected the fluorescence intensity of Bs2, so as to characterize the transcription of Bs2 regulated by Pfba promoter.

Experiment Results:

(1)Plasmid construction

Using the recombinant plasmid Pthl-Bs2 as template and Bs2-F and Bs2-R as primers, VBs2 vector (5664 bp) was amplified. Using Clostridium tyrobutyricum (C. tyrobutyricum) genome as template, Pfba gene fragment (300 bp) was amplified with Pfba-F and Pfba-R as primers. Gibson assembly method was used to link the Pfba fragment to the VBs2 linearized vector. Colony PCR (400 bp) was performed on the transformed colonies, using Bs2-PF and Bs2-PR as primers. The positive colonies were transferred and plasmid was extracted. After sequencing verification, the recombinant plasmid was obtained: pMTL-Pfba-Bs2.

Table 1 Primer sequences

(2)Fluorescence intensity

By using E. coli CA434 as a donor strain, pMTL-Pfba-Bs2 plasmid was transferred to C. tyrobutyricum (notated as Pfba). The transfected C. tyrobutyricum was cultured in RCM medium till OD600 reached 0.8 and 1.2, and detected for fluorescence intensity. C. tyrobutyricum transfected with empty vector pMTL82151 was used as blank control (notated as Pcontrol).

Fig. 2 Comparison of fluorescence intensity of C. tyrobutyricum transfected with pMTL-Pfba-Bs2 and that transfected with pMTL82151 empty vector

Our results showed that compared with the blank control (4720.67 and 1474.67), Pfba achieved efficient expression of Bs2 gene with strong fluorescence intensity of 33204.00 and 24397.00 under OD600=0.8 and OD600=1.2.

Contribution of NJTech-China-A 2023 team

(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, and photos under fluorescence microscopy.

In this study, we expressed the BS2 protein with the pET29a plasmid (containing the T7 promoter) (Fig. 1). To expand the application in facultative anaerob, we used the facultative anaerobe Escherichia coli strain BL21 as the expression vector to express the BS2 protein (Fig. 2). After 48 hours of cultivation, BS2 was fully released by sonication, and the excitation wavelength was measured to be approximately 447 nm, while the emission wavelength was approximately 521 nm using an multifunctional microplate detector (Fig. 3).

Fig. 1. Construction maps of plasmids of pET29a-BS2.


Fig. 2.Picture of solid medium under UV and fluorescence microscopic image. E. coli BL21 with pET29a-BS2 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 OD was ∼0.8.


Fig. 3.Excitation maximum and emission peak (RFU: Relative fluorescence unit). The Ex Wavelength in nm (Em: 520) indicates that there are two peak values of excite wavelength and the stronger one is 447 nm. The Em Wavelength in nm (Ex: 447 nm) shows excluding the impact of one peak value of excite wavelength, the emission wavelength is around 521 nm.

(2)The expression of BS2 protein in the facultative anaerobe

Based on the measured excitation/emission wavelengths, we controlled the cultivation temperature and time to measure the unit fluorescence intensity changes of E. coli BL21 with pET29a-BS2. After entering the logarithmic growth phase (OD600 ~0.5), IPTG was added to induce BS2 gene expression, and the OD600 and fluorescence intensity were measured every 20 minutes. Once in the steady phase, the OD600 and fluorescence intensity were measured every 1 hour. The unit fluorescence intensity of BS2 in E. coli BL21 was determined (Fig 3).

Fig. 4. RFU and RFU/OD600 under the growth curve of E. coli BL21. (A) is incubated at 20℃; (B) is incubated at 30℃. (C) is incubated at 37℃. (A)-(B) indicates that OD600 has the minimal fluctuation at 37℃, however at 30℃ MFI maintained the highest than the other two.

Contribution of NJTech-China-A 2024 team

Introduction
To optimize the expression of Bs2, we harnessed 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 without any addition of inducers, such as IPTG. 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). We 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.
Plasmid Construction
The recombinant plasmid pET29a-BS2 (including T7 promoter) was used as template, and j23119-For-20240719 and j23119-Rev-20240719 were used as primers to linearize vector (5695 bp) and one-step assembly was performed directly to construct PET29a-BS2 (J23119) (Fig 1). Using yanzheng23119-For and yanzheng23119-Rev as primers, colony PCR (734 bp) was performed on the transformed colonies. Positive colonies were transferred and plasmid was extracted. After sequencing verification, the recombinant plasmid PET29a-BS2 (J23119) was obtained.

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

Table 1 Primer sequences

Excitation maximum and emission peak of Bs2 in E.coli BL21(DE3)
Further, we employed the facultative anaerobic E. coli strain BL21(DE3) to express Bs2 in plasmid pET29a-Bs2 (J23119). The pictures of recombiant strains on the LB plate and fluorescence microscopic image indicated the successful expression of Bs2 (Fig 2). Following 48 hours of cultivation, the excitation wavelength of Bs2 expressed from the pET29a-Bs2 (J23119) recombinant plasmid was approximately 448 nm, with an emission wavelength around 509 nm, as measured by a multifunctional microplate detector (Fig 4).

Fig 2.E.coli BL21 with pET29a-Bs2 (J23119) on LB solid medium with kanamycin(10μg/ml) incubated in 37 ℃ for 24h. All was observed under UV.

Fig 2.A fluorescence microscopic image taken from the exponential growth phase when OD₆₀₀=0.8.

Fig.4 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.

Fluorescence intensity detection
The recombinant plasmid pET29-Bs2 (J23119) was introduced into Escherichia coli strain BL21(DE3) (notated as J23119). The transfected Escherichia coli was cultured in LB medium till OD₆₀₀ reached 0.8, 1.0 and 1.2, and the fluorescence intensity was detected. Escherichia coli BL21(DE3) transfected with pET29a empty vector as a blank control (notated as BL21). Compared with the blank control (261.97, 287.54, 304.34), J23119 achieved efficient expression of Bs2 gene with high RFU of 566.44, 1943.8 and 2767.61 under OD₆₀₀ of 0.8, 1 and 1.2 (Fig 5).

Fig.5 Comparison of RFU of Escherichia coli strain BL21(DE3) transfected with recombinant plasmid pET29-Bs2(J23119) and that transfected with pET29a empty vector (RFU: Relative fluorescence unit)

The optimization of cultivation temperature on Bs2 expression in E. coli BL21(DE3)
Finally, we adjusted cultivation temperature and time to evaluate changes in unit fluorescence intensity of E. coli BL21(DE3) with the pET29a-Bs2 (J23119) plasmid. 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). Fig 6 A-4C indicated that the recombiant strain showed better fluorescence intensity at 30℃ and 37℃.

Fig.6. 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.

Contribution of Ulink-SZ 2024 team

(1) Overview

Since T7 promoter is a promoter that needs to be induced by IPTG, and IPTG is expensive and contains slightly toxic, we designed a pET-P(BAD)-BS2 plasmid that only needs non-toxic arabinose to induce, and determined whether the pET-P(BAD)-BS2 plasmid could work normally in Escherichia coli by detecting the fluorescence expression intensity of fluorescent protein Bs2.

(2) Experiment Results

1.Plasmid construction

Recombinant plasmid pET29a-Bs2 (containing T7 promoter) was used as template, and Bs2-BAB-For and Bs2-BAB-Rev were used as primers to amplify Bs2 target gene (436bp). And pET-PBAD was used as template to amplified the arabinose-induced promoter. BAB-gj-For and BAB-gj-Rev were primer linearized vectors (5070bp) and linked to target genes by one-step cloning.

Fig. 1：pET-PBad-BS2

2.Fluorescence intensity detection

Our results showed that Escherichia coli BL21(DE)3 harboring pET-PBAD-BS2 achieved efficient expression of Bs2 gene with higher RFU of 428.52, 719.85 and 772.77 under OD600 of 0.8, 1, and 1.2, respectively, compared with the control (251.17, 267.38, 273.85).

Fig.2 Comparison of RFU of Escherichia coli strain BL21 transfected with recombinant plasmid pET-PBAD-Bs2 and that transfected with pET-PBAD empty vector. RFU: Relative fluorescence unit; BS2: Escherichia coli BL21(DE)3 harboring pET-P(BAD)-BS2; BL21: Escherichia coli BL21(DE)3 harboring pET-PBAD.

3.Excitation maximum and emission peak

In order to expand its application in facultative anaerobic bacteria, we used facultative anaerobic Escherichia coli strain BL21(DE)3 as expression cell to express Bs2 protein. After 48 hours of culture, the optimal excitation wavelength and the emission wavelength was detected (Fig.3). The Ex Wavelength in nm (Em: 520) indicates that there is one peak value of excite wavelength and it is 400 nm. The Em Wavelength in nm (Ex: 400 nm) shows excluding the impact of three peaks value of excite wavelength, the emission wavelength is around 525 nm.

Fig. 3 Excitation maximum and emission peak (RFU: Relative fluorescence unit).

4. 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(DE)3 introduced with pET-P(BAD)-BS2 plasmid by controlling the culture temperature and time. After entering the logarithmic growth phase (OD600~0.5), 10 mM arabinose was added to E. coli BL21(DE)3 harboring pET-P(BAD)-BS2 plasmid to induce Bs2 gene expression. OD600 and fluorescence intensity were measured every 1 h, and OD600 and fluorescence intensity were measured every 2 h after entering the stable phase. The unit fluorescence intensity of Bs2 in E. coli BL21(DE)3 was determined (Fig 4). (A)-(C) indicates that OD600 has the minimal fluctuation at 30℃. Besides, at 30℃, RFU maintained the highest than the other two.

Fig. 4 RFU and RFU/OD600 under the growth curve of E. coli harboring pET-P(BAD)-BS2 plasmid. (A) is incubated at 20℃; (B) is incubated at 30℃. (C) is incubated at 37℃.