Difference between revisions of "Part:BBa K4119002"
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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. | 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. | ||
| − | ==NJTech-China-A 2023 team contribution== | + | ==NJTech-China-A 2023 team <i><b>contribution</b></I>== |
===(1)Excitation maximum and emission peak=== | ===(1)Excitation maximum and emission peak=== | ||
Revision as of 10:03, 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
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 215
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 215
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 215
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 215
- 1000COMPATIBLE 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.
(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
(1)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 (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 enzyme- linked immunosorbent assay (ELISA) reader (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 (‰) and IPTG (10μL), 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. 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)Unit fluorescence intensity
Based on the measured excitation/emission wavelengths, we controlled the cultivation temperature and time to measure the unit fluorescence intensity changes of BS2 in E. coli growth curve. 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 growth curve was obtained (Fig 3).
Fig. 4. RFU and MFI 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.