TALANTA
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2025
·
IF 6.1
Tiantian Chen,Qian Xiang,Xuemei Sang,Daxiu Li,Wenjiao zhou,Fengfeng Xu
Malaria remains a significant threat to global health, especially with the emergence of drug-resistant strains. Therefore, precise monitoring of antimalarial drug levels is crucial for assessing and managing drug resistance. Here, we report a multiplex aptasensor that enables simultaneous fluorescence detection of two antimalarial drugs (MQ and PQ) via split dsT7 promoter-mediated transcription of two-color light-up RNA aptamers. The detection mechanism relies on specific binding of target antimalarial drugs (MQ/PQ) to their aptamers, each triggering formation of intact dsT7 promoter to induce amplified transcription of its cognate RNA aptamer. The generated RNA aptamers bind their corresponding organic dyes (TO1-Biotin and MG) with high affinity, yielding 7.7-/25-fold fluorescence enhancement to enable ultrasensitive detection of MQ and PQ with limits of 0.71 ng/mL and 0.069 ng/mL, respectively. The method also exhibits high selectivity and low background signal. Furthermore, this label-free system achieves multiplexed detection of antimalarial drugs in 10-fold diluted human serum (recovery: 98.67–106.84 % for PQ and 99.89–106.19 % for MQ), demonstrating its real utility. This label-free system is adaptable to other biomarkers through aptamer replacement.
研究领域:
生物化学
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分子生物学
·
医学诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Synthetic Biology
·
2025
·
IF 3.9
Hanqing Zhang,Luai R. Khoury,Peng Xu
Yarrowia lipolytica, a nonconventional yeast, has become an industrial workhorse to synthesize valuable compounds, including lipids, oleochemicals, and nutraceuticals. While the synthetic biology toolkits to engineer the genome and endogenous metabolic pathways are not as developed as Baker’s yeast, it has emerged as a promising microbial host for industrial applications. In this study, we examined the multiplexed editing capability of the CRISPR-AsCpf1 coupled with gRNAs generated from either a yeast native promoter or an orthogonal T7 promoter, which yielded 73.3% editing efficiency for up to four target genes and 100% editing efficiency for two genes. We also attempted two strategies to enhance homology-directed recombination (HDR) efficiency; only minor improvements were observed. We further demonstrated that CRISPR-dAsCpf1 with T7-driven gRNA achieved significant gene knockdown compared to the CRISPR-RfxCas13d system. Its knockdown efficiency was comparable to that of an antisense T7 promoter system. Taken together, this work provides a facile toolkit that enables efficient and multiplexed genome editing and transcriptional knockdown of critical genes by combining CRISPR-Cpf1 with an orthogonal T7 transcription system in Y. lipolytica.
研究领域:
合成生物学
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基因工程
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微生物生物技术
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分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2023
·
IF 8.4
Qing Han,Xinlan Bian,Yue Chen,Bingzhi Li
Flap endonuclease I (FEN1) is a structure-specific nuclease that participates in DNA repair, and its overexpression is related to an increased risk of cancer. Currently, few FEN1 assays can meet the requirements for diagnostic purposes due to their high complexity, low specificity, and limited sensitivity. Herein, we developed a biosensor adopting synthetic biology strategy, called CLAIM (cleavage-ligation-activation-involved monitoring) for FEN1 quantification. CLAIMintegrates ligation-based T7 promoter repair, RNA generation, and CRISPR/Cas13a. FEN1 cleavages the DNA probe with a 5′-flap to generate a nick site that can be ligated to form a T7 promoter. Subsequent transcription produces abundant RNA that can activate Cas13a, generating a fluorescence signal due to the trans -cleavage of Cas13a to a fluorogenic reporter. CLAIM was able to detect FEN1 with low femtomolar sensitivity, and its accuracy was validated using samples of cancer and non-tumorigenic cell lines. Interestingly, we found that the utilization of thermostable T4 DNA ligase is critical to make the cascade of FEN1 cleavage, ligation, and transcription take place in one-pot. CLAIM was also repurposed for validating FEN1 inhibitors, indicating its potential in drug screening. This work provides a diagnostic tool for FEN1 analysis and opens up opportunities for FEN1 biosensing and related drug discovery.
研究领域:
分子生物学
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合成生物学
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癌症诊断
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生物技术
✓ 已验证
DOI
高影响因子
Bioresources and Bioprocessing
·
2022
·Vol. 9
·期 1
·1-12
·
IF 4.983
Ye Jing,Li Yunjie,Bai Yuqing,Zhang Ting,Jiang Wei,Shi Ting,Wu Zijian,Zhang Yi-Heng P. Job
To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis. This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25–40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation.
研究领域:
微生物学
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生物技术
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分子生物学
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基因工程
✓ 已验证
DOI
PubMed
高影响因子
Synthetic and Systems Biotechnology
·
2025
·
IF 4.4
Yuke Qin,Shuting Hu,Yibin Yang,Xiaoqiang Jia
The research on whole-cell biosensors tailored for trace and ultra-trace detection remains limited and the biosensors based on natural bacterial heavy metal resistance mechanisms have common issues of low sensitivity. In this study, we designed a promoter P T7-cadO using the Cadmium ions (Cd 2+ )-binding protein binding site cadO with the T7 promoter at first and constructed a single-input whole-cell biosensor, which was named CP100, whose detection limit for Cd 2+ met the WHO requirement, yet its response and sensitivity were quite low. We further introduced the lacI and lac operator ( lacO ) as the signal amplifier to construct a dual-input promoter P T7-cadO-lacO-cadO and developed a biosensor named LC100, whose response and sensitivity were significantly improved, but background leakage became a new problem. Then we redesigned the gene circuit based on the regulatory circuit LCPM-2, which has the structure of “CadR-P J23100 -P T7-cadO-lacO-cadO -mRFP1-LacI”, with the LacI protein as the autoregulatory negative feedback model and finally obtained the biosensor named LC100-2, which achieved the detection of ultra-trace Cd 2+ (0.00001-0.02 nM), with the sensitivity of 3748.22 times that of CP100. Moreover, LC100-2 demonstrated excellent specificity to Cd 2+ among four other divalent metal ions and good anti-interference capability in the mixed divalent metal ions system. The results of the real water sample tests demonstrated that precise quantitative detection of Cd 2+ with a final concentration of 0.001-0.02 nM could be achieved by adding only a small volume of the sample (1 μL). This finding showed promising application potential in the field of trace detection. Additionally, the regulatory circuit LCPM-2 based on the unique dual-input promoter enhanced responses and reduced background leakage simultaneously, thereby providing an innovative strategy for the design and simplification of biosensors’ circuits.
研究领域:
生物传感器
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合成生物学
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环境监测
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分子生物学
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基因工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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