A self-amplifying plasmid based ultrasensitive biosensor for the detection of As(Ⅲ) in water

Li, Jiajia; Cui, Minghang; Zhao, Jing; Wang, Jin; Fang, Xiaona

doi:10.1016/j.bios.2022.114937

Cited by 12 publications

(5 citation statements)

References 54 publications

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“…Furthermore, a truncated repL sequence of 88 bp in size allowed RepL-mediated signal amplification in trans thus indicating a minimal sequence that could serve as RepL target site and contain all the DNA elements required for RepL-mediated plasmid DNA replication. Consistent with previous studies by Sheth et al (2017) , Dwidar and Yokobayashi (2019) , and Li et al (2023) , we have demonstrated that the RepL could amplify the signal of an arsenic biosensor. Furthermore, our data indicated that base substitution(s) at a single or several positions within the AT2 region produced varying levels of RepL-mediated signal amplification.…”

Section: Discussionsupporting

confidence: 92%

“…Therefore, RepL expression can be decoupled from the cis-acting plasmid amplifying effect, by introducing mutations to disrupt the oriL which is predicted to be located within repL gene sequence (Sheth et al, 2017). Consistently, Li et al (2023) demonstrated that RepL expression could increase the copy number of plasmids, which amplified the signal of an E. coli-based As(III) biosensor therefore indicating the potential use of such construct as a signal amplifier for E. coli cell-based genetic circuits.…”

Section: Introductionmentioning

confidence: 75%

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An adenine/thymidine-rich region is integral to RepL-mediated DNA replication

2023

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The lytic replication of bacteriophage P1 requires RepL expression and the lytic stage origin, oriL, which is postulated to be located within repL gene sequence. The exact sequence of P1 oriL and the mechanism(s) of RepL-mediated DNA replication, however, are not fully understood. By using repL gene expression to induce DNA replication of a gfp and a rfp reporter plasmids, we demonstrated that synonymous base substitution in an adenine/thymidine-rich region of repL gene sequence, termed AT2, significantly inhibited the RepL-mediated signal amplification. Contrastingly, mutations in an IHF and two DnaA binding sites did not affect the RepL-mediated signal amplification significantly. A truncated repL sequence with the AT2 region allowed RepL-mediated signal amplification in trans therefore verifying a significant role of the AT2 region in RepL-mediated DNA replication. A combination of repL gene expression and a non-protein-coding copy of repL gene sequence (termed nc-repL) was able to amplify the output of an arsenic biosensor. Furthermore, mutation(s) at single or multiple positions within the AT2 region produced varying levels of RepL-mediated signal amplification. Overall, our results provide novel insights into the identity and location of P1 oriL as well as demonstrating the potential of using repL constructs to amplify and modulate the output of genetic biosensors.

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Section: Discussionsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 75%

An adenine/thymidine-rich region is integral to RepL-mediated DNA replication

2023

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“…In addition to the accumulation of indigo, the concentration of As(III) did not affect bacterial growth (Figure b). No indigo background production was detected, outperforming reported biosensors. , The blue color signal remained stable after 8 h of As(III) induction in a dose and time-dependent manner, as shown in Figure c. No cytotoxicity was observed up to 150 μM As(III) (Figure d).…”

Section: Resultsmentioning

confidence: 84%

Biosynthesis of Indigo Dyes and Their Application in Green Chemical and Visual Biosensing for Heavy Metals

Guo,

Hu,

et al. 2024

ACS Omega

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Fermentative production of natural colorants using microbial strains has emerged as a cost-effective and sustainable alternative to chemical synthesis. Visual pigments are used as signal outputs in colorimetric bacterial biosensors, a promising method for monitoring environmental pollutants. In this study, we engineered four self-sufficient indigo-forming enzymes, including HbpAv, bFMO, cFMO, and rFPMO, in a model bacterium E. coli. TrxA-bFMO was chosen for its strong ability to produce indigo under T7 lac and mer promoters' regulation. The choice of bacterial hosts, the supplementation of substrate L-tryptophan, and ventilation were crucial factors affecting indigo production. The indigo reporter validated the biosensors for Hg(II), Pb(II), As(III), and Cd(II). The biosensors reported Hg(II) as low as 14.1 nM, Pb(II) as low as 1.5 nM, and As(III) as low as 4.5 nM but increased to 25 μM for Cd(II). The detection ranges for Hg(II), Pb(II), As(III), and Cd(II) were quantified from 14.1 to 225 nM, 1.5 to 24.4 nM, 4.5 to 73.2 nM, and 25 to 200 μM, respectively. The sensitivity, responsive concentration range, and selectivity are comparable to βgalactosidase and luciferase reporter enzymes. This study suggests that engineered enzymes for indigo production have great potential for green chemical synthesis. Additionally, heterologous biosynthesis of indigo production can lead to the development of novel, low-cost, and mini-equipment bacterial biosensors with zero background noise for visual monitoring of pollutant heavy metals.

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“…Controlled replication of plasmids is another method for safeguarding recombinant genes ( Li et al, 2023 ). RepL, a replication protein that primarily regulates plasmid copy number, was introduced to a plasmid as a plasmid amplifier.…”

Section: Microbial Detection and Remediation: Limitations And Current...mentioning

confidence: 99%

Synthetic bacteria for the detection and bioremediation of heavy metals

Thai

Lim

2023

Front. Bioeng. Biotechnol.

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Toxic heavy metal accumulation is one of anthropogenic environmental pollutions, which poses risks to human health and ecological systems. Conventional heavy metal remediation approaches rely on expensive chemical and physical processes leading to the formation and release of other toxic waste products. Instead, microbial bioremediation has gained interest as a promising and cost-effective alternative to conventional methods, but the genetic complexity of microorganisms and the lack of appropriate genetic engineering technologies have impeded the development of bioremediating microorganisms. Recently, the emerging synthetic biology opened a new avenue for microbial bioremediation research and development by addressing the challenges and providing novel tools for constructing bacteria with enhanced capabilities: rapid detection and degradation of heavy metals while enhanced tolerance to toxic heavy metals. Moreover, synthetic biology also offers new technologies to meet biosafety regulations since genetically modified microorganisms may disrupt natural ecosystems. In this review, we introduce the use of microorganisms developed based on synthetic biology technologies for the detection and detoxification of heavy metals. Additionally, this review explores the technical strategies developed to overcome the biosafety requirements associated with the use of genetically modified microorganisms.

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A self-amplifying plasmid based ultrasensitive biosensor for the detection of As(Ⅲ) in water

Cited by 12 publications

References 54 publications

An adenine/thymidine-rich region is integral to RepL-mediated DNA replication

An adenine/thymidine-rich region is integral to RepL-mediated DNA replication

Biosynthesis of Indigo Dyes and Their Application in Green Chemical and Visual Biosensing for Heavy Metals

Synthetic bacteria for the detection and bioremediation of heavy metals

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