A positive Selection Escherichia Coli Recombinant Protein Expression Vector for One-Step Cloning

We introduce OLIVAR (Orientation seLection of Insert in Vector through Antisense Reporter) as a novel selection strategy for the insertion of protein-coding genes into vector backbones. As a proof-of-concept, we have engineered a plasmid vector, pGRASS (Green fluorescent protein Reporter from Antisense promoter-based Screening System), for gene cloning in E. coli. With pGRASS, positive clones can be effortlessly distinguished from negative clones after blunt-end cloning. The vector not only screens clones with an insert but also for its correct orientation. The design further allows for the expression of recombinant protein from the T7 promoter in an appropriate host bacterium. With this vector, we are able to reduce the entire cloning workflow into a single step involving a 2-h reaction at room temperature. We believe that our cloning-cum-screening system presented here is extremely cost-effective and straightforward and can be applied to other vector systems and domains such as phage display and library construction.

Recombinant Production of Biliverdin IXβ and δ Isomers in the T7 Promoter Compatible Escherichia coli Nissle

The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXβ and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXβ and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOα) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXβ and -δ isomers >300-fold when compared to the chemical oxidation of heme.

Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor

Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Developing systems to induce localized sequence diversification at high efficiency will expand our ability to evolve traits of interest that improve global food security. In this study, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP-DNA base editor in Nicotiana benthamiana transient assays to target a transgene expressing GFP under the control of the T7 promoter. More than 7% of C nucleotides were converted to T in long segments of the GFP sequence. We then targeted the T7 promoter-driven ACETOLACTATE SYNTHASE (ALS) sequence that had been stably integrated into the rice (Oryza sativa) genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as a selection pressure for the evolution of the ALS sequence, resulting in the enrichment of herbicide-responsive residues. We then targeted these herbicide-responsive regions in the rice genome using a CRISPR-directed evolution platform and identified herbicide-resistant ALS variants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance, as well as for other trait engineering applications.

Construction of an AI-2 quorum sensing induced heterologous protein expression system in Escherichia coli

Background The pET expression system based on T7 promoter which is induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) is by far the most commonly used system for production of heterogeneous proteins in Escherichia coli. However, this system was limited by obvious drawbacks including the host toxicity and metabolic burden imposed by the presence of IPTG. Methods In this study, we incorporated the autoinducer-2 (AI-2) quorum sensing system to realize autoinduction of the pET expression system. The autoinduction expression vector pXWZ1 was constructed by inserting the lsr promoter regions into the pET28a(+) vector. The expression efficiency of the reporter genes gfpuv and lacZ by the pXWZ1 and pET28a(+) vectors were compared. Results The results showed that the expression levels of the both report genes in the cells transformed with pXWZ1 without any addition of exogenous inducer were higher than that transformed with pET28a(+) vectors by the induction of IPTG. Conclusion This new auto-induction system will exclude the limitations of the IPTG induction including toxic to host and increasing formation of inclusion body and will become a more economical and convenient tool for recombinant protein expression.

Building an RNA-based Toggle Switch using Inhibitory RNA Aptamers

Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in-vitro RNA-based toggle switch using RNA aptamers ca- pable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and Malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 in- hibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in cell-like con- ditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control ele- ment for nucleic acid networks in synthetic biology applications.

Characterization and reverse genetic establishment of cattle derived Akabane virus in China

Background Akabane virus (AKAV) is an important insect-borne virus which is widely distributed throughout the world except the Europe and is considered as a great threat to herbivore health. Results An AKAV strain defined as TJ2016 was firstly isolated from the bovine sera in China in 2016. Sequence analysis of the S and M segments suggested that the isolated AKAV strain was closely related to the AKAV strains JaGAr39 and JaLAB39, which belonged to AKAV genogroup II. To further study the pathogenic mechanism of AKAV, the full-length cDNA clone of TJ2016 S, M, and L segment was constructed separately into the TVT7R plasmid at the downsteam of T7 promoter and named as TVT7R-S, TVT7R-M, and TVT7R-L, respectively. The above three plasmids were further transfected into the BSR-T7/5 cells simultaneously with a ratio of 1:1:1 to produce the rescued virus AKAV. Compared with the parental wild type AKAV (wtAKAV), the rescued virus (rAKAV) was proved to be with similar cytopathic effects (CPE), plaque sizes and growth kinetics in BHK-21 cells. Conclusion We successfully isolated a AKAV strain TJ2016 from the sera of cattle and established a reverse genetic platform for AKAV genome manipulation. The established reverse genetic system is also a powerful tool for further research on AKAV pathogenesis and even vaccine studies.

Development and Characterization of a Reverse Genetics System for a Human-Derived Severe Fever With Thrombocytopenia Syndrome Virus Isolate From South Korea

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, tick-borne Bandavirus that causes lethal disease in humans. As there are no licensed vaccines and therapeutics for SFTSV, there is an urgent need to develop countermeasures against it. In this respect, a reverse genetics (RG) system is a powerful tool to help achieve this goal. Herein, we established a T7 RNA polymerase-driven RG system to rescue infectious clones of a Korean SFTSV human isolate entirely from complementary DNA (cDNA). To establish this system, we cloned cDNAs encoding the three antigenomic segments into transcription vectors, with each segment transcribed under the control of the T7 promoter and the hepatitis delta virus ribozyme (HdvRz) sequences. We also constructed two helper plasmids expressing the nucleoprotein (NP) or viral RNA-dependent RNA polymerase (RdRp) under the control of the T7 promoter and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). After co-transfection into BHK/T7-9 cells with three transcription and two helper plasmids, then passaging in Vero E6 or Huh-7 cells, we confirmed efficient rescue of the recombinant SFTSV. By evaluating the in vitro and in vivo virological properties of the parental and rescued SFTSVs, we show that the rescued virus exhibited biological properties similar to those of the parental virus. This system will be useful for identifying molecular viral determinants of SFTSV infection and pathogenesis and for facilitating the development of vaccine and antiviral approaches.

Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2

Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; these can be used to modify the ZIKV genomic RNA at the DNA level. A homologous recombination clone was used to construct pWSK29, a low copy plasmid that contained sequences for a T7 promoter, the whole genome of ZIKV ZKC2 strain, and a hepatitis delta virus ribozyme. High fidelity PCR was then used to amplify the T7 transcription template. The transcript was then transfected into susceptible cells via lipofection to recover the ZIKV ZKC2 strain. Finally, the virulence of rZKC2 was evaluated both in vitro and in vivo. The rZKC2 was successfully obtained and it showed the same virulence as its parent, the ZIKV ZKC2 strain (pZKC2), both in vitro and in vivo. The 3730 (NS2A-D62G) mutation site was identified as being important, since it had significant impacts on rZKC2 recovery. The 4015 (NS2A, A157V) mutation may reduce virus production by increasing the interferon type I response. In this study, one of the earliest strains of ZIKV that was imported into China was used for infectious clone construction and one possible site for antiviral medication development was discovered. The use of homologous recombination clones, of PCR products as templates for T7 transcription, and of lipofection for large RNA transfection could increase the efficiency of infectious clone construction. Our infectious clone provides an effective tool which can be used to explore the life cycle and medical treatment of ZIKV.

Modification Of Taq DNA Polymerase with Improved Elongation Ability

Thermostable polymerases play a significant role in molecular biology and diagnostic practice. The most famous and demanded is Polymerase I from the thermophilic bacterium Thermus aquaticus (Taq-pol). This polymerase at one time made a kind of revolution in the polymerase chain reaction. In this work, we attempted to modify this polymerase by attaching an additional Sso7d protein from Sulfolobus solfataricus to Taq-pol, which provides additional binding to the double-stranded DNA of the template. Sso7d-Taq fusion gene was expressed in BL21(DE3) cells. Optimal conditions were selected for maximum production of modified Sso7d-Taq polymerase. The optimal conditions for the intracellular accumulation of Sso7d-Taq polymerase: activation of the T7 promoter when the optical density of the culture reaches OD600 = 0.8-1.0 by adding IPTG at a concentration of 0.2 mM, followed by incubation of the culture at 37°C for 20-24 hours. Recombinant Sso7d-Taq polymerase has been purified and tested by PCR for thermal stability and elongation time. It was found that the Sso7d-Taq enzyme withstands 5 hour incubation at 95°C and 75 minute incubation at 98°C. Comparative analysis with unmodified Taq DNA polymerase showed that the Sso7d-Taq enzyme reduces the elongation rate by several times - up to 15-13 seconds per 1 kbp. The results obtained indicate the prospects of using Sso7d-Taq DNA polymerase in scientific research and diagnostic practice.

Highly Sensitive Luminescent Bioassay Using Recombinant Escherichia coli Biosensor for Rapid Detection of Low Cr(VI) Concentration in Environmental Water

In this study, we constructed a recombinant Escherichia coli strain with different promoters inserted between the chromate-sensing regulator chrB and the reporter gene luxAB to sense low hexavalent chromium (Cr(VI)) concentrations (<0.05 mg/L); subsequently, its biosensor characteristics (sensitivity, selectivity, and specificity) for measuring Cr(VI) in various water bodies were evaluated. The luminescence intensity of each biosensor depended on pH, temperature, detection time, coexisting carbon source, coexisting ion, Cr(VI) oxyanion form, Cr(VI) concentration, cell type, and type of medium. Recombinant lux-expressing E. coli with the T7 promoter (T7-lux-E. coli, limit of detection (LOD) = 0.0005 mg/L) had the highest luminescence intensity or was the most sensitive for Cr(VI) detection, followed by E. coli with the T3 promoter (T3-lux-E. coli, LOD = 0.001 mg/L) and that with the SP6 promoter (SP6-lux-E. coli, LOD = 0.005 mg/L). All biosensors could be used to determine whether the Cr(VI) standard was met in terms of water quality, even when using thawing frozen cells as biosensors after 90-day cryogenic storage. The SP6-lux-E. coli biosensor had the shortest detection time (0.5 h) and the highest adaptability to environmental interference. The T7-lux-E. coli biosensor—with the optimal LOD, a wide measurement range (0.0005–0.5 mg/L), and low deviation (−5.0–7.9%) in detecting Cr(VI) from industrial effluents, domestic effluents, and surface water—is an efficient Cr(VI) biosensor. This unprecedented study is to evaluate recombinant lux E. coli with dissimilar promoters for their possible practice in Cr(VI) measurement in water bodies, and the biosensor performance is clearly superior to that of past systems in terms of detection time, LOD, and detection deviation for real water samples.