Tianwen Wang

Available methods for assembling expression cassettes for synthetic biology

Studies in the structural biology of the multicomponent protein complex, metabolic engineering, and synthetic biology frequently rely on the efficient over-expression of these subunits or enzymes in the same cell. As a first step, constructing the multiple expression cassettes will be a complicated and time-consuming job if the classic and conventional digestion and ligation based cloning method is used. Some more efficient methods have been developed, including (1) the employment of a multiple compatible plasmid expression system, (2) the rare-cutter-based design of vectors, (3) in vitro recombination (sequence and ligation independent cloning, the isothermally enzymatic assembly of DNA molecules in a single reaction), and (4) in vivo recombination using recombination-efficient yeast (in vivo assembly of overlapping fragments, reiterative recombination for the chromosome integration of foreign expression cassettes). In this review, we systematically introduce these available methods.

Overview of Regulatory Strategies and Molecular Elements in Metabolic Engineering of Bacteria

From a viewpoint of biotechnology, metabolic engineering mainly aims to change the natural status of a pathway in a microorganism towards the overproduction of certain bioproducts. The biochemical nature of a pathway implies us that changed pathway is often the collective results of altered behavior of the metabolic enzymes encoded by corresponding genes. By finely modulating the expression of these genes or the properties of the enzyme, we can gain efficient control on the pathway. In this article, we reviewed the typical methods that have been applied to regulate the expression of genes in metabolic engineering. These methods are grouped according to the operation targets in a typical gene. The transcription of a gene is controlled by an indispensable promoter. By utilizing promoters with different strengths, expected levels of expression can be easily achieved, and screening a promoter library may find suitable mutant promoters that can provide tunable expression of a gene. Auto-responsive promoter (quorum sensing (QS)-based or oxygen-inducible) simplifies the induction process by driving the expression of a gene in an automated manner. Light responsive promoter enables reversible and noninvasive control on gene activity, providing a promising method in controlling gene expression with time and space resolution in metabolic engineering involving complicated genetic circuits. Through directed evolution and/or rational design, the encoding sequences of a gene can be altered, leading to the possibly most profound changes in properties of a metabolic enzyme. Introducing an engineered riboswitch in mRNA can make it a regulatory molecule at the same time; ribosomal binding site is commonly engineered to be more attractive for a ribosome through design. Terminator of a gene will affect the stability of an mRNA, and intergenic region will influence the expression of many related genes. Improving the performance of these elements are generally the main activities in metabolic engineering.

Anti-oxidative stress and beyond: multiple functions of the protein glutathionylation.

Glutathionylation, covalently attaching glutathione(s) to cysteine residue(s) of a protein, has attracted great attention in recent years. The importance of glutathionylation was initially recognized for its role in protecting proteins from irreversible oxidation; however, more studies indicate that glutathionylation is also involved in redox regulation under both normal physiological conditions and oxidative stresses. Potential mechanisms for the formation of glutathionylated proteins have been proposed. Despite the differences among the details of these mechanisms, glutathionylation is generally induced by intermediates including glutathione disulfide, protein-sulfenic acids, and thiyl radical. Taking advantages of proteomics techniques, authors have established methods to identify glutathionylation utilizing (35)S-cysteine- or biotin-labeled glutathione, or anti-GSH antibodies. Glutathionylation serves multiple roles in cellular biochemistry, such as modulation of enzymatic activity, glutathione storage, and dynamic regulation of protein function. Development of more efficient methods for glutathionylation identification, systematic investigation of its roles in the context of cellular biochemistry, the interaction with other types of protein modification, and its relevance to some health-threatening diseases will be the wider focus of studies in protein glutathionylation.

Screening and characterization of an aerobic nitrifying-denitrifying bacterium from activated sludge

Taking advantage of the good biocompatibility and high efficiency of nitrogen removal with microbes, nitrifying and denitrifying bacteria, are becoming increasingly more widely used for wastewater treatment and prevention of eutrophication. In this research, an aerobic nitrifying-denitrifying bacterium was successfully screened from activated sludge and identified as Pseudomonas sp. (CCTCC No M2010209) by the 16S rDNA sequence. The activity verification confirmed its nitrifying-denitrifying capability of removing ammonium, nitrate and nitrite nitrogen. The types of carbon sources and carbon-nitrogen ratio greatly influenced the removal efficiency of NH4+-N and NO3−-N. When the initial concentrations of NH4+-N and NO3−-N in synthetic wastewater were less than 70 and 50 mg/L, the nitrogen removal rates reached 94 and 90% in 9 h, respectively. Preliminary comparisons of nitrogen removal capacity between this isolate and other commercial preparations in the treatment of synthetic wastewater revealed its promising potential to be used in the actual wastewater treatment.

A Simple and Universal Method to Express Protein in Unfused form

The effect of additional amino acid residue(s) fused to the N- and/or C-terminal on properties of the heterogeneously expressed protein is usually difficult to be predicted. Recombinant proteins expressed without any fused sequence should be the most desired materials for related studies, such as protein drug preparation, biochemistry investigations. Here, we report a very simple and universal method enabling the expression of protein in its unfused form between the same two restriction enzyme sites (at a higher level) if a plasmid can support the fused expression. The method provided an assessable solution for unfused expression without increase in experimental resource; the necessary material is an additional primer. The method is especially useful for making whole-cell biocatalyst in which no purification steps are required.