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

(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.

Contribution of NanjingBioX 2024 team

(1) Overview

BS2 is one of the fluorescent proteins based on flavin mononucleotide (FMN). Currently, there is limited data on BS2 in the parts library. In order to apply it in practice, we have conducted a series of studies. We placed BS2 under the control of promoter Pi23100 and achieved efficient expression of this gene in the parthenogenetic anaerobic bacterium Escherichia coli in BL21 (DE3) in the absence of inducers. The measurement conditions of BS2 were optimized to determine its optimal excitation wavelength of 400 nm and emission wavelength of about 525 nm. Finally, we found that the fluorescent protein expression system performed best at 30 °C.

(2) Experiment Results

1.Plasmid construction

The recombinant plasmid pET29a-BS2 (including T7 promoter) was used as template, and j23100-For-20240719 and j23100-Rev-20240719 were used as primers to linearize vector(5721bp) and one-step cloning was performed directly. Using yanzheng23100-For and yanzheng23100-Rev as primers, colony PCR (734bp) was performed on the transformed colonies. Positive colonies were transferred and plasmid was extracted. After sequencing verification, the recombinant plasmid pET29a-Pj23100-BS2 was obtained.

Fig. 1：pET29a-Pj23100-BS2

2.Fluorescence intensity detection

In order to expand its application in facultative anaerobic bacteria, we used facultative anaerobic Escherichia coli strain BL21(DE)3 as the expression cell to express the BS2 protein. Subsequently, the recombinant plasmid pET29a-Pj23110-BS2 was introduced into Escherichia coli strain BL21(DE)3. The transfected Escherichia coli was cultured in LB medium until the OD600 reached 0.8, 1.0, and 1.2, and then the fluorescence intensity was detected. Meanwhile, Escherichia coli BL21(DE)3 transfected with the pET29a empty vector was used as a control.

Fig.2：Comparison of RFU of Escherichia coli strain BL21(DE)3 harboring recombinant plasmid pET29a-Pj23100-BS2 and pET29a empty vector. RFU: Relative fluorescence unit; J23100: Escherichia coli BL21(DE)3 harboring pET29a-Pj23100-BS2; BL21: Escherichia coli BL21(DE)3 harboring pET29a.

Our results showed that Escherichia coli BL21(DE)3 harboring pET29a-Pj23100-BS2 achieved efficient expression of BS2 gene with higher RFU of 332.53, 596.60 and 669.68 under OD600 of 0.8, 1, and 1.2, respectively compared with the control(261.97, 287.54, 304.34).

3.Exploring the optimal excitation and emission wavelengths for BS2

The fluorescence values of the engineered bacteria containing the recombinant plasmid were detected at different excitation and emission wavelengths after 48 hours of incubation. The results of the experiment are shown in Fig. The optimal excitation wavelength is 400 nm and the optimal emission wavelength is 525 nm after excluding the emission wavelength.

Fig. 3：Peak excitation and peak emission (RFU: Relative Fluorescence Unit).

4.Exploring the optimal expression temperature of BS2

According to the measured excitation/emission wavelength, we measured the change of unit fluorescence intensity of E. coli BL21(DE)3 harboring PET29a-Pj23100-BS2 by controlling the culture temperature and time. After entering the logarithmic growth phase (OD600~0.5), 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 was determined. The results showed that the fluctuation of OD600 was minimized at 37°C, but at 30°C, RFU remained at its highest value.

Fig. 4：RFU and RFU/OD600 under the growth curve of E. coli BL21(DE)3 harboring PET29a-Pj23100-BS2. (A) is incubated at 20℃; (B) is incubated at 30℃. (C) is incubated at 37℃.

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℃.