Supervisor(s): Chinese Academy of Sciences Sponsor(s): Institute of Microbiology, Chinese Academy of Sciences(CAS); Chinese Society for Microbiology CN：11-1998/Q
Founded in 1985,Chinese Journal of Biotechnology is the official journal of the Institute of Microbiology, the Chinese Academy of Sciences and the Chinese Society for Microbiology. It is an international,peer-reviewed journal that publishes original papers and reviews on all aspects of Biotechnology, such as genetic engineering,cell engineering, enzyme engineering, biochemical engineering, and so on.The journal is indexed/abstracted in various important citation resources such as Chemistry Abstracts,MEDLINE/PubMed,AJ OF vINITI, JST, Scopus, Center for Agriculture and Bioscience International, EMBASE, CNKI,CBM,Chinese Science and Technological Periodical(CD).
Efficient utilization of cellulose and xylan is of importance in the bioethanol industry. In this study, a novel bifunctional xylanase/cellulase gene,
Tcxyn10a, was cloned from
Thermoascus crustaceus JCM12803, and the gene product was successfully overexpressed in
Pichia pastoris GS115. The recombinant protein was then purified and characterized. The optimum pH and temperature of
TcXyn10A were determined to be 5.0 and 65 °C–70 °C, respectively. The enzyme retained stable under acid to alkaline conditions (pH 3.0–11.0) or after 1-h treatment at 60 °C. The specific activities of
TcXyn10A towards beechwood xylan, wheat arabinoxylan, sodium carboxymethyl cellulose, and lichenan were (1 480 ± 26) U/mg, (2 055 ± 28) U/mg, (7.4 ± 0.2) U/mg, and (10.9 ± 0.4) U/mg, respectively. Homologous modeling and molecular docking analyses indicated that the bifunctional
TcXyn10A had a single catalytic domain, in which the substrates xylan and cellulose shared the same binding cleft. This study provides a valuable material for the study of structure and function relationship of bifunctional enzymes.
Transcriptional regulation is crucial for regulated gene expression. Due to the complexity, it has been difficult to engineer eukaryotic transcription factor (TF) and promoter pairs. The few availabilities of eukaryotic TF and promotor pairs limit their practical use for clinical or industrial applications. Here, we reported a
de novo construction of synthetic inhibitory transcription factor and promoter pairs for mammalian transcriptional regulation. The design of synthetic TF was based on the fusion of DNA binding domain and Kruppel associated box transcription regulating domain (KRAB). The synthetic promoter was constructed by inserting the corresponding TF response element after SV40 promoter. We constructed and tested five synthetic inhibitory transcription factor and promoter pairs in cultured mammalian cells. The inhibition capability and orthogonality were verified by flow cytometry. In summary, we demonstrated the feasibility of constructing mammalian inhibitory TF and promoter pairs, which could be standardized for advanced gene-circuit design and various applications in the mammalian synthetic biology.
To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I + J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I + J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.
Various genetic switches have been developed to let engineered cells perform designed functions. However, a sustained input is often needed to maintain the on/off state, which is energy-consuming and sensitive to perturbation. Therefore, we developed a set of transcriptional switches for cell state control that were constructed by the inversion effect of site-specific recombinases on terminators. Such a switch could respond to a pulse signal and maintain the new state by itself until the next input. With a bottom-up design principle, we first characterized the terminators and recombinases. Then, the mutual interference was studied to select compatible pairs, which were used to achieve one-time and two-time state transitions. Finally, we constructed a biological seven-segment display as a demonstration to prove such switch’s immense potential for application.
Several putative transcription factor binding sites (TFBSs) exist in the
rep gene promoter of porcine circovirus type 2 (PCV2). To explore if PCV2 could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the
rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified that the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2
rep gene promoter. Dual-luciferase reporter assay, quantitative real-time PCR, Western blotting, and indirect immunofluorescent assay demonstrated that poTFAP2δ could not only promote the activity of the
rep gene promoter, but also enhance the transcription/translation activity of the rep/
cap gene and the virus titer of PCV2 during the entire life cycle of PCV2 infection. This study revealed the molecular mechanism of PCV2 using host proteins to enhance the viral replication, provided a new perspective for studying the pathogenic mechanism of PCV2 from virus and host interaction, and a theoretical basis for developing highly effective PCV2 vaccines.
Ebola virus (EBOV) is an extremely contagious pathogen first discovered in Africa associated with severe hemorrhagic disease in humans and nonhuman primates, which has resulted in at least 28 500 suspected cases and 11 300 confirmed deaths in 2014–2016 Ebola epidemic in West Africa. Rapid and sensitive detection of EBOV is the key to increasing the probability of survival and reducing infection rates in pandemic regions. Here, we report an ultrasensitive and instrument-free EBOV detection assay based on colloidal carbon immunochromatography. Carbon nanoparticle-labeled rabbit anti-EBOV-VP40 IgG were concentrated in the conjugate pad, monoclonal antibody (McAb, 4B7F9) against EBOV-VP40 and goat anti-rabbit IgG were immobilized on the nitrocellulose membrane with 2 μL/cm at a concentration of 1 mg/mL as test and control lines, respectively. Then the sample application pad, conjugate release pad, nitrocellulose membrane and absorbent pad were assembled into a lateral flow test strip. The test strip shows strong specificity against related viruses that share similar clinical symptoms and geographic range with EBOV, including Marburg virus, influenza virus, yellow fever virus and dengue virus. In addition, 1 500 negative serums were tested with false-positive rate of 1.3‰ which significantly lower than that of ReEBOV™ colloidal gold test kit recommended by World Health Organization (WHO). The sensitivity of this strip was analyzed using inactivated EBOV with detection limit of 100 ng/mL (10
6 copies/mL) which clearly higher than that of ReEBOV™ dipstick (10 μg/mL, 10
8 copies/mL). Furthermore, the strip showed excellent thermal stability characteristics in room temperature and could be as a point-of-care (POC), ultra-sensitive and specific promising candidate for EBOV serological screening in rural Africa or entry/exit ports.
Genetically engineered intestinal microbes could be powerful tools for the detection and treatment of intestine inflammation due to their non-invasive character, low cost, and convenience. Intestinal inflammation is usually detected along with an increasing concentration of thiosulfate and tetrathionate molecules in the intestines. ThsSR and TtrSR are two-component biosensors to detect the presence of thiosulfate and tetrathionate molecules, respectively. In real-life intestinal inflammation detection, sophisticated instruments are needed if fluorescent proteins are used as reporters. However, chromoproteins and other colored small molecules, which can be observed by the unaided eye, could extend the use of ThsSR and TtrSR biosensors to detect intestine inflammation. The feasibility of ThsSR and TtrSR systems was tested by monitoring the fluorescence intensity of sfGFP in response to the concentrations of thiosulfate and tetrathionate, followed by the incorporation of the two systems into
Escherichia coli Top10 and
E. coli Nissle 1917. Considering the application in real-life, sfGFP was substituted by a series of chromoproteins and chromogenic small molecules. The potential for the real-life application of the two systems was further corroborated by substituting sfGFP with a series of chromoproteins and a protoviolaceinic acid synthesis cassette as reporter genes in
E. coli Top10. The results indicated that signal expression of the new systems had a positive correlation with the concentration of tetrathionate or thiosulfate. Thus, the modified ThsSR and TtrSR systems may potentially be applied in human body for the detection of intestinal inflammation in families.
