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</p> <p>Author: Yuyao Cao, Yijiu Lu</p> <p><b>Summary: Characterization of the transcription of Bs2 gene regulated by Pfba promoter <p>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.