Use of the redox-dependent intein system for enhancing production of the cyclic green fluorescent protein.

To expand the reported redox-dependent intein system application, in this work, we used the split intein variant with highly trans-splicing efficiency and minimal extein dependence to cyclize the green fluorescent protein variant reporter in vitro. The CPG residues were introduced adjacent to the intein's catalytic cysteine for reversible formation of a disulfide bond to retard the trans-splicing reaction under the oxidative environment. The cyclized reporter protein in Escherichia coli cells was easily prepared by organic extraction and identified by the exopeptidase digestion. The amounts of extracted cyclized protein reporter in BL21 (DE3) cells were higher than those in hyperoxic SHuffle T7 coexpression system for facilitating the disulfide bond formation. The double His6-tagged precursor was purified for in vitro cyclization of the protein for 3 h. Compared with the purified linear counterpart, the cyclic reporter showed about twofold increase in fluorescence intensity, exhibited thermal and hydrolytic stability, and displayed better folding efficiency in BL21 (DE3) cells at the elevated temperature. Taken together, the developed redox-dependent intein system will be used for producing other cyclic disulfide-free proteins. The cyclic reporter is a potential candidate applied in certain thermophilic aerobes.

Establishment of an efficient seed fluorescence reporter-assisted CRISPR/Cas9 gene editing in maize.

Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR-associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter-assisted CRISPR/Cas systems can greatly facilitate the selection of genome-edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)-assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En-SFR/Cas9), embryos (Em-SFR/Cas9), or both tissues (Em/En-SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En-SFR/Cas9, Em-SFR/Cas9, and Em/En- SFR/Cas9 to identify plants not harboring the genome-editing cassette but carrying the desired mutations at target genes in single genes or in small-scale mutant libraries, and report on the successful generation of single-target mutants and/or mutant libraries with En-SFR/Cas9, Em-SFR/Cas9, and Em/En-SFR/Cas9. SFR-assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.

Three-dimensional automated reporter quantification (3D-ARQ) technology enables quantitative screening in retinal organoids.

The advent of stem cell-derived retinal organoids has brought forth unprecedented opportunities for developmental and physiological studies, while presenting new therapeutic promise for retinal degenerative diseases. From a translational perspective, organoid systems provide exciting new prospects for drug discovery, offering the possibility to perform compound screening in a three-dimensional (3D) human tissue context that resembles the native histoarchitecture and to some extent recapitulates cellular interactions. However, inherent variability issues and a general lack of robust quantitative technologies for analyzing organoids on a large scale pose severe limitations for their use in translational applications. To address this need, we have developed a screening platform that enables accurate quantification of fluorescent reporters in complex human iPSC-derived retinal organoids. This platform incorporates a fluorescence microplate reader that allows xyz-dimensional detection and fine-tuned wavelength selection. We have established optimal parameters for fluorescent reporter signal detection, devised methods to compensate for organoid size variability, evaluated performance and sensitivity parameters, and validated this technology for functional applications.

Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus.

BACKGROUND: Genetic transformation of microalgae has been hampered by inefficient transgene expression, limiting the progress of microalgal biotechnology. Many vector tools and strategies have been developed in recent years to improve transgene expression in the model microalga Chlamydomonas, but these were hardly applied to other microalgae. In this work, naturally-isolated oleaginous microalgae were accessed for genetic transformation, and various expression systems were evaluated in a selected microalga to circumvent inefficient transgene expression. RESULTS: Initially, a strain of Scenedesmus acutus was selected from the oleaginous microalgal collection based on its highest transformation rate and transgene stability. This strain, which had very low or no GFP reporter expression, was first tested to improve transgene expression by using intron-containing constructs and the transcript fusion using ble::E2A. The intron-containing constructs yielded 2.5-7.5% of transformants with 2-4-fold fluorescence signals, while the majority of the transformants of the transcript fusion had the fluorescence signals up to 10-fold. Subsequently, three UV-induced S. acutus mutants were isolated with moderate increases in the level and frequency of transgene expression (2-3-fold and 10-12%, respectively). Finally, a transcript fusion system was developed using psy white mutants with an expression vector containing PSY::E2A for complementation and light selection. Transformants with green colonies were selected under light exposure, and the transgene expression was detected at protein levels. Although the improvement using PSY::E2A was only minor (1-2-fold increase and ~ 7% of transformants), this system provides an alternative selectable marker that is compatible with large-scale culture. CONCLUSIONS: Here, the overall improvement of transgene expression using the Chlamydomonas tools was moderate. The most effective tool so far is the transcript fusion using ble::E2A system. This work demonstrates that, so far, genetic engineering of non-model microalgae is still a challenging task. Further development of tools and strategies for transgene expression in microalgae are critically needed.

U13 → C13 mutation in the variable region of the NA gene 3'UTR of H9N2 influenza virus influences the replication and transcription of NA and enhances virus infectivity.

The untranslated regions within viral segments are the essential promoter elements required for the initiation of viral replication and transcription. The end of the UTR sequence and part of the ORF sequence constitute the packaging signal for progeny viruses. To explore the influence of single-point and multi-site joint mutations in the UTR of the NA gene on the viral expression, we select clones with upregulated expression of the reporter gene and analyze their sequence characteristics. Bioinformatics methods were used to analyze polymorphisms in the untranslated region (UTR) of the neuraminidase gene of the H9N2 influenza A virus. Using the RNA polymerase I reporting system with enhanced green fluorescence protein (EGFP) gene as the reporter gene, libraries containing random mutations at sites within the N2 UTR were constructed using random mutagenesis. The mutants were selected from the randomized mutagenesis libraries for the N2-UTR. The N2-UTR-RNA polymerase I fluorescence reporter system was identified by sequencing and transfected into infected MDCK cells. The expression of the reporter EGFP was observed using fluorescence microscopy, and the relative fluorescence intensity was measured using a multifunctional microplate reader to analyze the expression of the reporter gene (EGFP) qualitatively and quantitatively. Herein, an RNA polymerase reporter system was constructed to rescue the mutated viruses and measure their tissue culture infective dose (TCID50). The results showed that the U13 → C13 mutation in the 3'end of the NA gene promoted the expression of viral RNA and protein, and mutation of other sites within the UTR could differentially regulate viral genomic transcription and translation. These data showed that the U13 → C13 mutation within the variable region of the 3'UTR of the NA gene in the H9N2 influenza virus promotes viral genomic expression and infection.

A Thioacetal Photocage Designed for Dual Release: Application in the Quantitation of Therapeutic Release by Synchronous Reporter Decaging.

Despite the immense potential of existing photocaging technology, its application is limited by the paucity of advanced caging tools. Here, we report on the design of a novel thioacetal ortho-nitrobenzaldehyde (TNB) dual arm photocage that enabled control of the simultaneous release of two payloads linked to a single TNB unit. By using this cage, which was prepared in a single step from commercial 6-nitroverataldehyde, three drug-fluorophore conjugates were synthesized: Taxol-TNB-fluorescein, Taxol-TNB-coumarin, and doxorubicin-TNB-coumarin, and long-wavelength UVA light-triggered release experiments demonstrated that dual payload release occurred with rapid decay kinetics for each conjugate. In cell-based assays performed in vitro, dual release could also be controlled by UV exposure, resulting in increased cellular fluorescence and cytotoxicity with potency equal to that of unmodified drug towards the KB carcinoma cell line. The extent of such dual release was quantifiable by reporter fluorescence measured in situ and was found to correlate with the extent of cytotoxicity. Thus, this novel dual arm cage strategy provides a valuable tool that enables both active control and real-time monitoring of drug activation at the delivery site.

Fluorescence reporting of G-quadruplex structures and modulating their DNAzyme activity using polyethylenimine-pyrene conjugate.

Four-stranded G-quadruplex structure is one of the most important non-canonical secondary structures of DNA formed by guanine (G)-rich sequences. G-rich DNA sequences are known to occur in the human genome, especially in the telomere 3' end and in oncogene promoters such as c-MYC and c-KIT. In this context, we designed pyrene-conjugated polyethylenimine (PEI-Py) as a fluorescence reporter for the recognition and detection of G-quadruplex structures of G-rich deoxyoligonucleotides and human telomere and gene promoter sequences, under ambient conditions. PEI-Py exhibited prominent pyrene excimer emission in the presence of G-quadruplex structures of G-rich deoxyoligonucleotides and biologically relevant DNA sequences. PEI-Py further displayed the modulation of DNAzyme activity of various G-quadruplex structures in the presence of hemin and hydrogen peroxide.

A fluorimetric readout reporting the kinetics of nucleotide-induced human ribonucleotide reductase oligomerization.

Human ribonucleotide reductase (hRNR) is a target of nucleotide chemotherapeutics in clinical use. The nucleotide-induced oligomeric regulation of hRNR subunit α is increasingly being recognized as an innate and drug-relevant mechanism for enzyme activity modulation. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides, hRNR-α assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger α-dimerization. Currently, both knowledge of and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. We therefore developed a fluorimetric assay that reliably reports on oligomeric state changes of α with high sensitivity. The oligomerization-directed fluorescence quenching of hRNR-α, covalently labeled with two fluorophores, allows for direct readout of hRNR dimeric and hexameric states. We applied the newly developed platform to reveal the timescales of α self-assembly, driven by the feedback regulator dATP. This information is currently unavailable, despite the pharmaceutical relevance of hRNR oligomeric regulation.

Construction of Infectious Clones for Human Enteroviruses.

The infectious clone has been constructed for years via various mechanisms using reverse genetics of viral RNA into cDNA. The mechanism of construction has evolved to DNA-launch plasmids which simplify infectious clone manipulation and expression in mammalian cells. Infectious clones have enormously allowed manipulation of the enterovirus genome to discover antivirals, viral replication mechanisms, and functions of essential viral proteins. Here we will be discussing methods for the production of DNA-launch human enterovirus infectious clones and viral genome engineered with a fluorescent reporter gene.

Production of the biocommodities butanol and acetone from methanol with fluorescent FAST-tagged proteins using metabolically engineered strains of Eubacterium limosum.

BACKGROUND: The interest in using methanol as a substrate to cultivate acetogens increased in recent years since it can be sustainably produced from syngas and has the additional benefit of reducing greenhouse gas emissions. Eubacterium limosum is one of the few acetogens that can utilize methanol, is genetically accessible and, therefore, a promising candidate for the recombinant production of biocommodities from this C1 carbon source. Although several genetic tools are already available for certain acetogens including E. limosum, the use of brightly fluorescent reporter proteins is still limited. RESULTS: In this study, we expanded the genetic toolbox of E. limosum by implementing the fluorescence-activating and absorption shifting tag (FAST) as a fluorescent reporter protein. Recombinant E. limosum strains that expressed the gene encoding FAST in an inducible and constitutive manner were constructed. Cultivation of these recombinant strains resulted in brightly fluorescent cells even under anaerobic conditions. Moreover, we produced the biocommodities butanol and acetone from methanol with recombinant E. limosum strains. Therefore, we used E. limosum cultures that produced FAST-tagged fusion proteins of the bifunctional acetaldehyde/alcohol dehydrogenase or the acetoacetate decarboxylase, respectively, and determined the fluorescence intensity and product concentrations during growth. CONCLUSIONS: The addition of FAST as an oxygen-independent fluorescent reporter protein expands the genetic toolbox of E. limosum. Moreover, our results show that FAST-tagged fusion proteins can be constructed without negatively impacting the stability, functionality, and productivity of the resulting enzyme. Finally, butanol and acetone can be produced from methanol using recombinant E. limosum strains expressing genes encoding fluorescent FAST-tagged fusion proteins.

A fluorescence-based high-throughput screening method for cytokinin translocation mutants.

BACKGROUND: Cytokinins are one kind of phytohormones essential for plant growth, development and stress responses. In the past half century, significant progresses have been made in the studies of cytokinin signal transduction and metobolic pathways, but the mechanism of cytokinin translocation is poorly understood. Arabidopsis (Arabidopsis thaliana) response regulator 5 (ARR5) is a type-A response factor in cytokinin signaling which is induced by cytokinins and has been used as a reporter gene for the endogenous cytokinins in Arabidopsis. Here, we report a fluorescence-based high-throughput method to screen cytokinin translocation mutants using an ethyl methyl sulfone (EMS) mutagenesis library generated with ARR5::eGFP transgenic plants. RESULTS: The seedlings with enhanced green fluorescent protein (GFP) signal in roots were screened in a luminescence imaging system (LIS) in large scale to obtain mutants with over-accumulated cytokinins in roots. The selected mutants were confirmed under a fluorescence microscopy and then performed phenotypic analysis. In this way, we obtained twelve mutants with elevated GFP signal in the roots and further found three of them displayed reduced GFP signal in the aerial tissues. Two of the mutants were characterized and proved to be the atabcg14 allelic mutants which are defective in the long-distance translocation of root-synthesized cytokinins. CONCLUSIONS: We provide a strategy for screening mutants defective in cytokinin translocation, distribution or signaling. The strategy can be adapted to establish a system for screening mutants defective in other hormone transporters or signaling components using a fluorescence reporter.

Design, cloning and characterization of transcription factor-based inducible gene expression systems.

Cellular functions are often controlled by small molecular weight molecules such as metabolites. Microorganisms, mainly prokaryotes, have evolved sensing and regulatory mechanisms based on transcriptional regulators (TRs) that are able to activate gene expression in response to changes in intra- and extracellular metabolite (ligand) concentrations. To understanding control mechanisms and cell factory development in synthetic biology applications, high throughput analytical procedures are required. In this chapter, we outline a methodological pipeline to design and build reporter constructs enabling the characterization of metabolite-responsive inducible gene expression systems. As an example, we present the design, cloning and characterization of the itaconate-inducible system which is composed of the LysR-type transcriptional regulator ItcR and the promoter Pccl from Yersinia pseudotuberculosis. Fluorescence-based plate reader and flow cytometry assays are described and the steps for performing data analysis are provided.

Development of an oxygen-independent flavin mononucleotide-based fluorescent reporter system in Clostridium beijerinckii and its potential applications.

Clostridium beijerinckii is a predominant solventogenic clostridia with great attraction for renewable liquid biofuel and biochemical production. Metabolic engineering and synthetic biology can be employed to engineer the strain toward desirable phenotypes. However, current limited information such as promoter strength and gene regulation may hinder the efficient engineering of the strain. To investigate genetic information and complex cellular bioprocesses of C. beijerinckii, an in vivo fluorescence reporter system can be employed. In general, green fluorescence protein (GFP) and relative analogs have been widely used as real-time reporters. However, GFP-family proteins require molecular oxygen for fluorescence maturation. Considering the strict anaerobic growth requirement of the clostridia, an oxygen-independent fluorescence reporter such as a flavin mononucleotide-based fluorescent protein (FbFP) can be used as an alternative fluorescence reporter. In this study, we synthesized and expressed the codon-optimized FbFP gene for C. beijerinckii (CbFbFP) based on the nucleotide sequence of Bacillus subtilis YtvA variant EcFbFP in C. beijerinckii NCIMB 8052 wild-type. Protein expression and in vivo fluorescence of CbFbFP in C. beijeirnckii were confirmed under anaerobic growth conditions. Through fluorescence-activated cell sorting (FACS), we isolated the bright cells from the heterogenous population of C. beijerinckii cells expressing CbFbFP. Several mutations were found in the isolated plasmid which may be responsible for the high-level expression of CbFbFP in C. beijerinckii. The mutant plasmid and CbFbFP reporter were further utilized for strain selection, real-time fluorescence measurement, population analysis, and metabolic engineering in this study.

SaCas9 Requires 5'-NNGRRT-3' PAM for Sufficient Cleavage and Possesses Higher Cleavage Activity than SpCas9 or FnCpf1 in Human Cells.

CRISPR/Cas9-mediated gene therapy holds great promise for the treatment of human diseases. The protospacer adjacent motif (PAM), the sequence adjacent to the target sequence, is an essential targeting component for the design of CRISPR/Cas9-mediated gene editing. However, currently, very few studies have attempted to directly study the PAM sequence in human cells. To address this issue, the authors develop a dual fluorescence reporter system that could be harnessed for identifying functional PAMs for genome editing endonuclease, including Cas9. With this system, the authors investigate the effects of different PAM sequences for SaCas9, which is small and has the advantage of allowing in vivo genome editing, and found only 5'-NNGRRT-3' PAM could induced sufficient target cleavage with multi-sites. The authors also found SaCas9 possesses higher activity than SpCas9 or FnCpf1 via plasmids (episomal) and chromosomes with integrated eGFP-based comparison. Taken together, the authors show that a dual fluorescence reporter system is a means to identifying a functional PAM and quantitatively comparing the efficiency of different genome editing endonucleases with the similar or identical target sequence in human cells.

HIVGKO: A Tool to Assess HIV-1 Latency Reversal Agents in Human Primary CD4+ T Cells.

While able to suppress HIV replication in HIV infected individuals, combination antiretroviral therapy (ART) fails to eliminate viral latent reservoir, which consists in integrated transcriptional silenced HIV provirus. So far, identification of latently-infected cells has relied on activating cells to induce expression of HIV proteins which can then be detected. Unfortunately, this activation significantly changed the cell phenotype. We developed a novel HIV reporter, named HIVGKO, that allows the purification of latently-infected cells in absence of reactivation. Indeed, latent cells can be identified by expression of the EF1a-driven mKO2 and lack of expression of the LTR-driven csGFP. This protocol can be used to study the effectiveness of LRAs (Latency Reversal Agents) in reactivating latent HIV in primary cells.

A dual fluorescence reporter system for high throughput screening of effectors of Kiss1 gene expression.

Kisspeptin is a multifunctional peptide encoded by the Kiss1 gene that plays critical roles in mammalian puberty onset modulation and fertility maintenance in the hypothalamus. Understanding how Kiss1 expression is regulated is essential for elucidating the molecular mechanisms responsible for these reproductive events. In this study, we constructed an in vitro dual fluorescence reporter system to facilitate high throughput screening of effectors influencing the expression of Kiss1. In GT1-7 cells, an enhanced GFP gene was placed under the control of the Kiss1 gene regulatory elements and translated together with this gene. A tdTomato gene cassette was simultaneously introduced into the same cell for normalization of the fluorescence signal. After treatment with different effectors, the cells were analyzed by flow cytometry. We first tested the efficacy of the system using canonical regulators and then carried out high throughput functional screening to identify chemical compounds that can regulate Kiss1 gene expression. Of 22 tested compounds from natural sources, 13 significantly affected Kiss1 expression. Verification by western blot and quantitative reverse transcription PCR (qRT-PCR) assays and structural analysis identified two chalcone compounds as possible regulators of Kiss1 gene expression. This system may be suitable for gene functional analysis, drug screening and pharmaceutical studies.

Quantitative Analysis of Intracellular Response Regulator Phosphatase Activity of Histidine Kinases.

Quantitation of two-component protein activities is becoming increasingly important to understand the general design principles for this widely distributed prokaryotic signaling pathway. In many two-component systems (TCSs), phosphatase activity of the sensor histidine kinase (HK) plays a major role in controlling the system output and resetting the system to the prestimulus state. Quantitation of the phosphatase activity is often carried out in vitro, usually with truncated proteins that may not recapitulate the intact HK in the cellular environment. This chapter outlines a method for characterizing the intracellular phosphatase activity by investigating the TCS deactivation dynamics upon stimulus withdrawal. Two experimental approaches, the direct Phos-tag gel analysis and the indirect reporter assay, are described for measuring the TCS deactivation dynamics in cell. Combined with a mathematic model, the experimentally determined kinetics can lead to proper evaluation of the intracellular phosphatase activity.

Dual Fluorescence Reporter Based Analytical Flow Cytometry for miRNA Induced Regulation in Mammalian Cells.

MicroRNA-induced gene regulation is a growing field in basic and translational research. Examining this regulation directly in cells is necessary to validate high-throughput data originated from RNA sequencing technologies. For this several studies employ luciferase-based reporters that usually measure the whole cell population, which comes with low resolution for the complexity of the miRNA-induced regulation. Here, we provide a protocol using a dual-fluorescence reporter and flow cytometry reaching single cell resolution; the protocol contains a simplified workflow that includes: vector generation, data acquisition, processing, and analysis using the R environment. Our protocol enables high-resolution measurements of miRNA induced post-transcriptional gene regulation and combined with system biology it can be used to estimate miRNAs proficiency.

Simple and rapid determination of homozygous transgenic mice via in vivo fluorescence imaging.

Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study.