A standardized genome architecture for bacterial synthetic biology (SEGA)

Chromosomal recombinant gene expression offers a number of advantages over plasmid-based synthetic biology. However, the methods applied for bacterial genome engineering are still challenging and far from being standardized. Here, in an attempt to realize the simplest recombinant genome technology imaginable and facilitate the transition from recombinant plasmids to genomes, we create a simplistic methodology and a comprehensive strain collection called the Standardized Genome Architecture (SEGA). In its simplest form, SEGA enables genome engineering by combining only two reagents: a DNA fragment that can be ordered from a commercial vendor and a stock solution of bacterial cells followed by incubation on agar plates. Recombinant genomes are identified by visual inspection using green-white colony screening akin to classical blue-white screening for recombinant plasmids. The modular nature of SEGA allows precise multi-level control of transcriptional, translational, and post-translational regulation. The SEGA architecture simultaneously supports increased standardization of genetic designs and a broad application range by utilizing well-characterized parts optimized for robust performance in the context of the bacterial genome. Ultimately, its adaption and expansion by the scientific community should improve predictability and comparability of experimental outcomes across different laboratories.

Generation of Fluorescent Bacteria with the Genes Coding for Lumazine Protein and Riboflavin Biosynthesis

Lumazine protein is a member of the riboflavin synthase superfamily and the intense fluorescence is caused by non-covalently bound to 6,7-dimethyl 8-ribityllumazine. The pRFN4 plasmid, which contains the riboflavin synthesis genes from Bacillus subtilis, was originally designed for overproduction of the fluorescent ligand of 6,7-dimethyl 8-ribityllumazine. To provide the basis for a biosensor based on the lux gene from bioluminescent bacteria of Photobacterium leiognathi, the gene coding for N-terminal domain half of the lumazine protein extending to amino acid 112 (N-LumP) and the gene for whole lumazine protein (W-LumP) from P. leiognathi were introduced by polymerase chain reaction (PCR) and ligated into pRFN4 vector, to construct the recombinant plasmids of N-lumP-pRFN4 and W-lumP-pRFN4 as well as their modified plasmids by insertion of the lux promoter. The expression of the genes in the recombinant plasmids was checked in various Escherichia coli strains, and the fluorescence intensity in Escherichia coli 43R can even be observed in a single cell. These results concerning the co-expression of the genes coding for lumazine protein and for riboflavin synthesis raise the possibility to generate fluorescent bacteria which can be used in the field of bio-imaging.

Fusion Expression and Fibrinolytic Activity of rPA/SP-B

Background: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). Objective: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied. Methods: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method. Results: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity. Conclusion: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.

Development of fluorescent protein-marked strains of Bacillus subtilis

We obtained constructs pHT01-GFP and pHT43-RFP. Recombinant plasmids were transferred into endophytic Bacillus subtilis (strains 10-4 and 26D).

The Combination of Molecular Adjuvant CCL35.2 and DNA Vaccine Significantly Enhances the Immune Protection of Carassius auratus gibelio against CyHV-2 Infection

Cyprinid herpesvirus 2 (CyHV-2) infection results in huge economic losses in gibel carp (Carassius auratus gibelio) industry. In this study, we first constructed recombinant plasmids pcORF25 and pcCCL35.2 as DNA vaccine and molecular adjuvant against CyHV-2, respectively, and confirmed that both recombinant plasmids could be effectively expressed in vitro and in vivo. Then, the vaccination and infection experiments (n = 50) were set as seven groups. The survival rate (70%) in ORF25/CCL35.2 group was highest. The highest specific antibody levels were found in ORF25/CCL35.2 group in major immune tissues by qRT-PCR, and confirmed in serum by ELISA assay, antibody neutralization titer, and serum incubation-infection experiments. Three crucial innate immune indices, namely C3 content, lysozyme, and total superoxide dismutase (TSOD) activities, were highest in ORF25/CCL35.2 group in serum. pcORF25/pcCCL35.2 can effectively up-regulate mRNA expressions of some important immune genes (IL-1β, IL-2, IFN-γ2, and viperin), and significantly suppress CyHV-2 replication in head kidney and spleen tissues. The minimal tissue lesions can be seen in ORF25/CCL35.2 group in gill, spleen, and trunk kidney tissues by histopathological examination. The results indicated that the combination of DNA vaccine pcORF25 and molecular adjuvant pcCCL35.2 is an effective method against CyHV-2 infection, suggesting a feasible strategy for the control of fish viral diseases.

Statistical noise from recombinant plasmids can be abated via complementation of a ribosomal protein gene deletion

The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, ‘ribosome pegging’, to exclude common unwanted mutations that benefit host cells at the expense of heterologous gene expression. Plasmids that constitutively co-expressed the fluorescent reporter tagRFP and ribosomal protein L23 (rplW) were used to transform Escherichia coli cells that lacked the essential chromosomal rplW gene. Cells within the population that expressed too little L23, or too much, were evidently inviable. Ribosome pegging obviates the need for antibiotics, thus facilitating the deployment of recombinant bacteria in uncontrolled environments. We show that ribosome-pegged E. coli carrying a plasmid that constitutively expresses L23 and an artificially evolved enzyme protects fruit flies from otherwise toxic doses of the insecticide malathion.

USING OF UNIVERSAL PLASMID CONSTRUCTIONS FOR DESIGN OF RECOMBINANT ANTIBODIES WITH DEFINED SPECIFICITY IN EUKARYOTIC CELLS

Aim. In this study we aimed to develop the methodology to change the antigen specificity of chimeric antibodies by replacing the variable region genes in the previously designed universal plasmid constructions pLK DT-17 and pHG DT-17 encoding the DT-17 antibody against the diphtheria toxin (DT) to the genes of antibody binding to another DT epitope — DT-22.Materials and methods. The genes of the light and heavy chain variable regions of mouse anti-DT antibodies — DT-22 were amplified from the hybridoma producing monoclonal antibodies to DT by reverse transcription and PCR methods. Genetic engineering methods were used to replace the variable regions of DT-17 antibody in the recombinant plasmids pLK DT-17 and pHG DT-17 encoding the light and heavy chains of DT-17 antibody, respectively to the relevant genes of DT-22. Subsequently, a «supervector» pSV DT-22, containing the genes of both chains of the chimeric antibody, was designed. CHO cells were transfected with a «supervector» and a highly productive clone, secreting chimeric antibodies to DT was obtained. Immunochemical and cultural methods were used to evaluate antibody activity. The affinity chromatography was used to purified preparative amounts of antibodies.Results. The yield of purified secreted chimeric DT-22 antibodies was 4 mg from per liter of culture medium. The minimum concentration of chimeric antibodies at which DT was neutralized in the CHO cells was 22 μg/mL of medium.Conclusion. Thus it has been shown how to generate new vector coding synthesis of light and heavy chains of a chimeric DT-22 antibody specific to another DT epitope using previously constructed universal recombinant plasmids pLK DT-17 and pHG DT-17 encoding, light and heavy chains of antibodies against DT DT-17, respectively.

In silico Designed Ebola Virus T-Cell Multi-Epitope DNA Vaccine Constructions Are Immunogenic in Mice

Background: The lack of effective vaccines against Ebola virus initiates a search for new approaches to overcoming this problem. The aim of the study was to design artificial polyepitope T-cell immunogens––candidate DNA vaccines against Ebola virus and to evaluate their capacity to induce a specific immune response in a laboratory animal model. Method: Design of two artificial polyepitope T-cell immunogens, one of which (EV.CTL) includes cytotoxic and the other (EV.Th)––T-helper epitopes of Ebola virus proteins was carried out using original TEpredict/PolyCTLDesigner software. Synthesized genes were cloned in pcDNA3.1 plasmid vector. Target gene expression was estimated by synthesis of specific mRNAs and proteins in cells transfected with recombinant plasmids. Immunogenicity of obtained DNA vaccine constructs was evaluated according to their capacity to induce T-cell response in BALB/c mice using IFNγ ELISpot and ICS. Results: We show that recombinant plasmids pEV.CTL and pEV.Th encoding artificial antigens provide synthesis of corresponding mRNAs and proteins in transfected cells, as well as induce specific responses both to CD4+ and CD8+ T-lymphocytes in immunized animals. Conclusions: The obtained recombinant plasmids can be regarded as promising DNA vaccine candidates in future studies of their capacity to induce cytotoxic and protective responses against Ebola virus.

Molecular cloning of enolase from Trichinella spiralis and the protective immunity in mice

Trichinella spiralis, the main pathogen of trichinosis, infects a wide range of mammalian hosts and is one of the most widespread parasites worldwide. For parasites, glycolysis is the most important way to generate energy. Previous studies showed that some enzymes involved in the glycolytic pathway play roles in regulation the host immunity. In this paper, enolase from T. spiralis was cloned and the protective potentials were studied. One hundred and sixty ICR mice were divided into four groups and vaccinated with recombinant enolase (pET-ENO), eukaryotic recombinant plasmid encoding enolase (pVAX1-ENO) and negative controls (pVAXl and PBS), respectively. Two weeks after the second immunization, each mouse was challenged orally with 200 muscle larvae (MLs) of T. spiralis. Results showed that mice vaccinated with pET-ENO and pVAX1-ENO induced specific antibodies of IgG, IgA, IgM, but no IgE. Subclasses of IgG antibodies showed that mice immunized with recombinant protein and recombinant plasmids induced a Th1/Th2 immune response. Concentrations of serum cytokines were detected and showed significant increase of IFN-γ, IL-4 and TGFβ1, while IL-17 in each group was not significantly different. Flow cytometric analysis showed significant increase of CD4+ and CD8+ T lymphocytes in the groups immunized with recombinant protein and recombinant plasmids. Challenge infection demonstrated that immunized groups had a reduced number of worm burdens. The reductions of larvae per gram muscle (LPG) in pET-ENO and pVAX1-ENO group were 17.7% and 15.8% when compared with PBS control.