Amino Acid Substitution ; Exocytosis ; HEK293 Cells ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Membrane Proteins ; chemistry ; metabolism ; Microscopy, Confocal ; Protein Binding ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; Sirolimus ; analogs & derivatives ; chemistry ; metabolism ; Tacrolimus Binding Proteins ; chemistry ; genetics ; metabolism

Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S. cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S. cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S. cerevisiae. The well-characterized DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S. cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of DsRed-Monomer gene. The successful construction of pGBT9Red lays the foundation for further analysis of promoter activity and screening of promoter element libraries.
Amino Acid Sequence ; Base Sequence ; genetics ; Cloning, Molecular ; DNA Primers ; biosynthesis ; Gene Expression Regulation, Fungal ; Genetic Vectors ; Luminescent Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Synthetic Biology ; Transformation, Genetic

OBJECTIVETo establish a bioluminescent MDA-MB-231 cell line which can stably express luciferase and green fluorescent protein to allow bioluminescent imaging in nude mouse models bearing human triple-negative breast cancer xenografts.

METHODSThe lentivirus carrying luc2, eGFP and neo fusion genes were packaged in 293T cells via calcium phosphate co-precipitation. Human triple-negative breast cancer cell line MDA-MB-231 was infected by the lentivirus, and the positive cell clones were tested for eGFP and luc2 expressions by fluorescence microscopy and Xenogen IVIS200 bioluminescent imaging system, respectively. MTT assay, transwell invasion assay and wound healing assay were performed to evaluate the changes in the proliferation, invasion and migration abilities of the infected cells. The cells were then orthotopically implanted into the right second mammary fat pat of female BALB/c nude mice. The tumor growth was monitored by the in vivo imaging system every week, and the tumor tissues were harvested to evaluate the in vivo stability and tumorigenicity of the modified cells using cryosection and HE staining.

RESULTSThe lentivirus-infected MDA-MB-231cells could stably express luc2 and eGFP, and the luciferase activity reached 9689 phontons/s/per cell. No significant changes occurred in the biological activities of the lentivirus-infected MDA-MB-231 cells. We successfully established the nude mouse model bearing orthotopically implanted human triple-negative breast cancer cells.

CONCLUSIONThe modified MDA-MB-231 cell line can be detected sensitively at the primary implantation site and distant metastasis site in nude mice, which provides a convenient and sensitive platform for the research of metastatic mechanism and new antitumor drugs of human triple-negative breast cancer. The combination of eGFP and luc2 is superior to single reporter gene.

Animals ; Brain Neoplasms ; secondary ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Genes, Reporter ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Luciferases ; biosynthesis ; genetics ; Luminescent Measurements ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism

Glutamate-1-semiadhyde aminotransferase (GSAT) is an enzyme in the upstream biosynthetic pathway of uroporphyrinogen III that is the substrate of uroporphyrinogen III methyltransferase (UPMT), a novel red fluorescent protein. In order to detect the effect of overexpression of GSAT with UPMT on the fluorescent intensity in Escherichia coli, we amplified maize upmt gene by PCR and inserted into the first cistron of pET Duet-1 plasmid to create the vector pETU. The expressed UPMT was fused histidine tag at N terminus. We also amplified E. coli hemL gene encoding GSAT by PCR reaction, eliminated Nco I site within the hemL gene by site-directed mutagenesis and subcloned into pET-51b plasmid. The resultant hemL gene was inserted the second cistron of pETU plasmid to produce the vector pETeGU. The expressed GSAT has the extra Strep-TagII at N terminus. Compared to overexpression upmt gene alone, coexpression both genes did not resulted in the remarkable change in either the amount of the UPMT, as estimated by western blot analysis, or the constitution of red fluorescent materials, as shown by UV/visible light scanning analysis, but increased cellular level of the fluorescent material trimethylpyrrocorphin with the specific absorption at 354 nm. The red fluorescence emitted by the colonies cooverexpressing both enzymes completely disappeared after treated by 2 mmol/L gabaculine, the GSAT inhibitor, suggested that the recombinant GSAT may increase the cellular level of uroporphyrinogen III, and thus enhanced the red fluorescence of the E. coli cells conferred by the recombinant UPMT.
Escherichia coli ; genetics ; metabolism ; Genes, Plant ; Genetic Vectors ; genetics ; Intramolecular Transferases ; biosynthesis ; genetics ; Luminescent Proteins ; biosynthesis ; genetics ; Methyltransferases ; biosynthesis ; genetics ; Mutagenesis, Site-Directed ; Recombinant Proteins ; biosynthesis ; genetics ; Zea mays ; genetics

The plasmodium of Physarum polycephalum is a suitable eukaryotic cell for cell cycle investigation, but there is no compatible transient expression system for the plasmodium. Using the promoter and terminator of ardC actin of Physarum polycephalum substituted the CMV IE and SV40 polyA of plasmid pDsRedl-N1, using cassette PardC-MCS-DsRed1-TardC substituted the cassette PardC-hph-TardC of plasmid pTB38, we constructed plasmids pXM1 and pXM2 for transient expression of red fluorescent protein (RFP) in Physarum polycephalum respectively. After reconstituting the transcription elongation factor homologous gene (pelf1) of Physarum polycephalum into the pXM2, we generated a plasmid pXM2-pelf1. After the plasmid pXM1, pXM2 and pXM2-pelf1 were electroporated into the plasmodium of Physarum polycephalum, we observed optimum RFP and PELF1-RFP expression under fluoroscope and confocal microscope between 24-48 h after electroporation, and found that ELF1-RFP expression was accumulated in nucleus of microplasmodium, the optimum electroporation parameters were 40 V/cm electric field, 1 ampere current, and 70 micros electric shock time. The results suggest that this expression system is qualified for transient expression of specific protein in plasmodium of Physarum polycephalum.
Actins ; genetics ; metabolism ; Electroporation ; Luminescent Proteins ; biosynthesis ; genetics ; Physarum polycephalum ; genetics ; metabolism ; Plasmids ; genetics ; metabolism ; Transcriptional Elongation Factors ; genetics

Based on the character of strong promoter of the fibroin gene and high level secretion of fibroin of Bombyx mori, we amplified the promoter of heavy chain gene (Fib-H) and its downstream signal peptide sequence (FibHS) by PCR. After that, we cloned the PCR product in pBluescriptII SK (+) to form the vector pSK-FibHS and analyzed its sequence. The sequence identity was 99% comparable to that of the reported sequence by Blast on line. Then we digested pSk-Ser-DsRed-PolyA with Sal IKpn I to get DsRed-PolyA DNA fragment and subcloned it into vector pSK-FibHS to generate a transitorily secretory expression vector pSK-FibHS-DsRed-PolyA. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pSK-FibHS-DsRed-PolyA into BmN cells by liposome. From the cells transfected with the recombinant vector, what the red fluorescence could be detected verified that the recombinant vector could express DsRed in BmN cells transiently. Furthermore, when silkworm had been injected with the recombinant vector pSK-FibHS-DsRed-PolyA, red fluorescence could be observed in the lumen of silk gland of silkworm. The result indicated that DsRed expressed transiently and was secreted into lumen of the silk gland. Therefore, we supposed that the cloned sequence (FibHS) possessed signal peptide bio-function. Moreover, this study would lay a foundation for the research on secretory expression of exogenous gene by silk gland bioreactor.
Amino Acid Sequence ; Animals ; Base Sequence ; Bombyx ; genetics ; metabolism ; Cloning, Molecular ; Fibroins ; biosynthesis ; genetics ; Insect Proteins ; biosynthesis ; genetics ; Luminescent Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics

We cloned genes of four sentrin-specific protease (SENP), three small ubiquitin-like modifiers (SUMO), enhanced cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) by two-step PCR. Then we constructed expression vector B28 for SENP and B13 for ECFP-SUMO-EYFP. The recombinant plasmids were transformed into Escherichia coli BL21 and expression was induced by isopropyl beta-D-thiogalactoside, then recombinant proteins were purified by Ni-NTA agarose column ion-exchange chromatography. The proteins were analyzed with SDS-PAGE and identified with Western blotting. Except that SENP3 catalytic domain (SENP3C) truncated in the C termini and SENP5C expressed in inclusion body, others were expressed as soluble proteins. SDS-PAGE analysis showed that the relative molecular mass of these fusion proteins were consistent with theoretical ones, and the specificity of the fusion proteins were confirmed with Western blotting. The fusion proteins of SENP and ECFP-SUMO-EYFP can be successfully expressed in prokaryotic expression system. It lays the foundation for the fluorescence resonance energy transfer analysis.
Bacterial Proteins ; biosynthesis ; genetics ; Catalytic Domain ; Cysteine Endopeptidases ; biosynthesis ; genetics ; Endopeptidases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Luminescent Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; SUMO-1 Protein ; biosynthesis ; genetics

OBJECTIVETo construct a red fluorescent protein reporter gene driven by human catalase gene promoter.

METHODSThe red fluorescent protein reporter gene plasmid pDsRed-CATp containing human catalase gene promoter was constructed by gene recombination technique. The plasmid was transiently transfected into NIH/3T3 cells to observe their response to H(2)O(2) stimulation.

RESULTSThe plasmid was constructed correctly as verified by double enzyme digestion and sequence analysis. The plasmid was lowly expressed in resting NIH/3T3 cells, but the expression level increased obviously after stimulation by H(2)O(2). CONCLSIONS: A red fluorescent protein reporter gene plasmid driven by human catalase gene promoter has been constructed successfully with a sensitive response to H(2)O(2) stimulation. This system provides a convenient tool for the study of the regulatory mechanism of catalase gene expression.

3T3 Cells ; Animals ; Catalase ; genetics ; Gene Expression ; Genes, Reporter ; genetics ; Genetic Vectors ; biosynthesis ; genetics ; Humans ; Hydrogen Peroxide ; pharmacology ; Luminescent Proteins ; biosynthesis ; genetics ; Mice ; Plasmids ; genetics ; Promoter Regions, Genetic ; Transfection

In order to trace the expression of exogenous hepatocyte growth factor (HGF) in eukaryotic cells exactly, a recombinant plasmid that expresses the fusion protein of HGF and red fluorescent protein (RFP) was constructed. The gene encoding HGF without stop code was amplified from pMD19-T-HGF by PCR technique and then cloned in pDsRed-express-N1. The recombinant plasmid (pDsRed-HGF) was identified by restriction endonuclease enzyme analysis and DNA sequence analysis. pDsRed-HGF was transfected into 293T cells. The expression of HGF mRNA, HGF and red fluorescent protein was detected. The results showed that the target gene sequence in pDsRed-HGF was completely in conformity with HGF cDNA in GenBank, and it was expressed highly in 293T cells. In this study, pDsRed-HGF was successfully constructed and expressed in eukaryotic cells.
Cell Line ; Genetic Vectors ; genetics ; Hepatocyte Growth Factor ; biosynthesis ; genetics ; Humans ; Luminescent Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

The regulation of a target gene expression is very important in gene therapy. However, constitutive or inappropriate expression of the genes with traditional expression system may interfere with the effect of the gene therapy, even may lead to lethal side effect. We constructed an RU486 inducible eukaryotic vector carrying DsRed protein and evaluated its regulatable effect in vitro. The single vector named PDC-RURED was constructed with molecular biological methods, which contained DsRed gene, promoter and RU486-inducible system. To minimize any potential interference, we spaced the two transcriptional elements with a 1.6 kb insulator. The vector was identified by different enzyme restrictions, sequencing analysis and PCR assay. We demonstrated the regulatable expression of this vector after transfection in HEK293 cells by fluorescence microscopy and flow cytometry. In the absence of RU486, no significant DsRed protein activation was observed, whereas in the presence of RU486 up to 40 fold activation of the DsRed protein was observed in cultured cells. The data show that the novel eukaryotic expression plasmid vector can be used to regulate the expression level of genes of interest in appropriate time under the control of RU486. This inducible expression vector provides a powerful tool for the research of gene regulation and gene therapy.
Cell Line ; Fluorescent Dyes ; metabolism ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Kidney ; cytology ; Luminescent Proteins ; biosynthesis ; genetics ; Mifepristone ; pharmacology ; Promoter Regions, Genetic ; genetics ; Transfection