Fluorescence-activated Cell Sorting Technology Research Articles

A Novel Method to Screen Strong Constitutive Promoters in Escherichia coli and Serratia marcescens for Industrial Applications.

Promoters serve as the switch of gene transcription, playing an important role in regulating gene expression and metabolites production. However, the approach to screening strong constitutive promoters in microorganisms is still limited. In this study, a novel method was designed to identify strong constitutive promoters in E. coli and S. marcescens based on random genomic interruption and fluorescence-activated cell sorting (FACS) technology. First, genomes of E. coli, Bacillus subtilis, and Corynebacterium glutamicum were randomly interrupted and inserted into the upstream of reporter gene gfp to construct three promoter libraries, and a potential strong constitutive promoter (PBS) suitable for E. coli was screened via FACS technology. Second, the core promoter sequence (PBS76) of the screened promoter was identified by sequence truncation. Third, a promoter library of PBS76 was constructed by installing degenerate bases via chemical synthesis for further improving its strength, and the intensity of the produced promoter PBS76-100 was 59.56 times higher than that of the promoter PBBa_J23118. Subsequently, promoters PBBa_J23118, PBS76, PBS76-50, PBS76-75, PBS76-85, and PBS76-100 with different strengths were applied to enhance the metabolic flux of L-valine synthesis, and the L-valine yield was significantly improved. Finally, a strong constitutive promoter suitable for S. marcescens was screened by a similar method and applied to enhance prodigiosin production by 34.81%. Taken together, the construction of a promoter library based on random genomic interruption was effective to screen the strong constitutive promoters for fine-tuning gene expression and reprogramming metabolic flux in various microorganisms.

Isolation and Characterization of Bone Marrow Mesenchymal Stromal Cell Subsets in Culture Based on Aldehyde Dehydrogenase Activity.

Mesenchymal stromal cells (MSCs) have particular properties that allow their use as therapeutic strategies for several cell-based applications. Historically, bone marrow (BM)-MSCs are isolated by culture adherence since specific cell surface markers are yet to be developed. This original work aimed to identify and characterize isolating expanded BM-MSCs based on their aldehyde dehydrogenase (ALDH) activity known to be a hallmark of stem cells and relevant for their isolation. We thus isolated by fluorescence-activated cell sorting technology two functionally different populations of BM-MSCs depending on their ALDH activity (ALDH+ and ALDH-). Transcriptome analysis and profiling clearly demonstrated that both populations of BM-MSCs present distinct pattern of genes related to the main properties of MSCs (proliferation, response to hypoxia, angiogenesis, phenotype, stemness, multilineage, hematopoiesis, immunomodulation) in an ALDH activity dependent manner. Both BM-MSC populations look to significantly differ in terms of biological responses and functionalities. More functional analyses are needed to understand and characterize the properties of these ALDH populations. Collectively, our results highlight ALDH activity as a potential feature for isolating and segregating functional and/or competent subset of BM-MSC populations, which may account for better and more efficient therapeutic issue.

Developing a high-throughput screening method for threonine overproduction based on an artificial promoter.

Backgroundl-Threonine is an important amino acid for animal feed. Though the industrial fermentation technology of threonine achieved a very high level, there is still significant room to further improve the industrial strains. The biosensor-based high-throughput screening (HTS) technology has demonstrated its powerful applications. Unfortunately, for most of valuable fine chemicals such as threonine, a HTS system has not been established mainly due to the absence of a suitable biosensor. In this study, we developed a HTS method to gain high-yielding threonine-producing strains.ResultsNovel threonine sensing promoters including cysJp and cysHp were discovered by proteomic analyses of Escherichia coli in response to extracellular threonine challenges. The HTS method was constructed using a device composed of the fused cysJp and cysHp as a promoter and a linked enhanced green fluorescent protein gene as a reporter. More than 400 strains were selected with fluorescence activated cell sorting technology from a library of 20 million mutants and tested within 1 week. Thirty-four mutants have higher productivities than the starting industrial producer. One mutant produced 17.95 % more threonine in a 5-L jar fermenter.ConclusionsThis method should play a functional role for continuous improvement of threonine industry. Additionally, the threonine sensor construction using promoters obtained by proteomics analyses is so convenient that it would be easily extended to develop HTS models for other biochemicals.

FACS technology used in a new rapid bacterial detection method

Culture methods for bacterial detection (BacT/ALERT or Pall eBDS) are currently implemented in blood donor screening procedures in many countries. Experience in the first years after implementation of these detection assays showed that although the analytical sensitivity was extremely high (about 1 CFU mL(-1)), the majority of these platelets were still transfused before a positive screening result was attained. Rapid technologies were developed to more effectively prevent transfusion-transmitted bacterial infection. In this study, a new rapid bacterial detection method based on fluorescence-activated cell sorting (FACS) technology was developed. Bacteria were stained with thiazole orange dye for 5 min and measurement was taken immediately after staining. The entire process took only 30 min. Six transfusion-relevant bacteria strains were tested in a spiking study. Without pre-incubation in a special bacteria growth medium, analytical sensitivity ranged between 10(5) CFU mL(-1) (Klebsiella oxytoca and Serratia marcesens) and 10(3) CFU mL(-1) (Escherichia coli). Sensitivity could be improved to 10(1) CFU mL(-1) for all tested bacteria by adding a pre-incubation step (6 h at 37 degrees C). Although preliminary in nature, results of our study suggest that bacterial detection by FACS technology in conjunction with a pre-incubation step offers a sensitive alternative technology to culture methods. Additionally, it provides the benefit of a rapid test time and the opportunity of preventing bacterial transmitted infections more effectively.

Presenilin-dependent Processing and Nuclear Function of γ-Protocadherins

The recently described protocadherin gene clusters encode cadherin-related proteins, which are highly expressed in the vertebrate nervous system. Here, we report biochemical studies addressing proteolytic processing of gamma-protocadherins. These type-I transmembrane proteins are cleaved by a metalloproteinase in vivo, generating a soluble extracellular fragment and a carboxyl-terminal fragment associated with the cellular membrane. In addition, we show that the carboxyl-terminal fragment is a substrate for further cleavage mediated by presenilin. Consequently, accumulation of the fragment is found when gamma-secretase is inactivated either by the specific presenilin-inhibitor L685,458 or in double mutant murine embryonic fibroblasts lacking both presenilin genes. The gamma-secretase-generated carboxyl-terminal fragment is largely unstable but accumulates when proteasomal degradation is inhibited. Interestingly, the proteolytic fragment generated by gamma-secretase can localize to the nucleus. This is the first report providing experimental evidence for a cell surface receptor signaling function of protocadherins regulated by proteolytic events.

Molecular pathways of monocyte emigration into the alveolar air space of intact mice.

The adhesive interactions involved in monocyte recruitment to the alveolar space in vivo are only poorly defined. To study these interactions, we used a recently developed mouse model that allowed the separation and quantification of freshly recruited monocytes, resident alveolar macrophages (rAM), neutrophils, and lymphocytes in the bronchoalveolar compartment by fluorescence activated cell sorting technology. In these mice, the combined intratracheal administration of the monocyte chemoattractant JE/monocyte chemotactic protein (MCP)-1 and low dose Escherichia coli lipopolysaccharide (LPS) induces a self-limiting pulmonary inflammatory response, characterized by well-controlled sequelae of both neutrophil and monocyte emigration into the alveolar space. In contrast, challenge with JE/MCP-1 provokes the emigration only of monocytes in the absence of lung inflammation. Using an array of function-blocking monoclonal antibodies (mAb) (anti-CD11a, -CD11b, -CD18, -CD49d, -CD54, and -CD106), we characterized the adhesive interactions underlying the transendothelial and transepithelial leukocyte traffic in intact animals. Alveolar monocyte recruitment elicited by JE/MCP-1 alone was strictly dependent on CD11b/CD18, CD54, and CD49d, and partly dependent on CD11a, but not dependent on CD106. In response to JE/MCP-1 plus E. coli LPS, we observed additional engagement of CD11a and CD106 for enhanced alveolar monocyte transmigration. Comigrating neutrophils were found to primarily utilize CD11b, CD18, and CD54, but not CD49d, CD106, or, surprisingly, CD11a. This contrasted with the effect of CD11a on alveolar challenge with macrophage inflammatory protein (MIP)-1alpha instead of JE/MCP-1. In conclusion, we found that in an intact mouse model allowing detailed phenotyping of leukocyte traffic into the alveolar space, the molecular pathways involved in JE/MCP-1-driven monocyte efflux differed under noninflammatory and inflammatory (presence of LPS) conditions. Moreover, the profile of adhesive interactions underlying the monocyte efflux differed from that characterizing neutrophil trafficking.