Difference between revisions of "Part:BBa K4119002"

Revision as of 08:34, 11 October 2023

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

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.

(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).

NJTech-China-A 2023 team contribution

Excitation maximum and emission peak

In this study, we constructed the BS2 gene with the pET29a plasmid (containing the T7 promoter) (Fig. 1). For simplification purposes, we used the facultative anaerobic E. coli strain BL21 as the expression vector to express the BS2 protein. 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 enzyme- linked immunosorbent assay (ELISA) reader (Fig. 2).

Fig. 1. Construction maps of plasmids of pET29a-BS2. Fig. 4. 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.