Design of recombinase and terminator-based genetic switches for cell state control / 生物工程学报

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 states 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.

Development of a novel vector for cloning and expressing extremely toxic genes in Escherichia coli

Background: Escherichia coli has been widely used as a host to clone and express heterologous genes. However, there are few vectors available for cloning and expressing extremely toxic genes, which limits further basic and applied research on extremely toxic proteins. Results: In this study, a novel vector pAU10 was constructed in E. coli. pAU10 utilizes the combination of the efficient but highly repressible T7-lacO promoter/operator and the strong rrnBT2 transcriptional terminator upstream of the T7 promoter to strictly control unwanted transcription of the extremely toxic gene; in addition, the trp promoter/operator is oriented opposite to the T7 promoter to control the production of the antisense RNA that may block the translation of leaky mRNA. Without the supplementation of IPTG and L-tryptophan in the culture medium, transcription of the extremely toxic gene by the T7 promoter is highly repressed, and the trp promoter produces the antisense RNA, which strictly prevents unwanted expression of the extremely toxic protein in E. coli. With the supplementation of IPTG and L-tryptophan, the T7 promoter efficiently transcribes the extremely toxic gene, and the trp promoter does not produce the antisense RNA, ensuring efficient expression of the extremely toxic protein in E. coli. Tight regulation and efficiency of expression of an extremely toxic gene cloned in the vector pAU10 were confirmed by cloning and expressing the restriction endonuclease-encoding gene bamHI without its corresponding methylase gene in E. coli JM109(DE3). Conclusion: pAU10 is a good vector used for cloning and expressing extremely toxic genes in E. coli.

Molecular cloning, expression, and immobilization of glutamate decarboxylase from Lactobacillus fermentum YS2

Background: GABA (γ-aminobutyric acid) is a four-carbon nonprotein amino acid that has hypotensive, diuretic, and tranquilizing properties. Glutamate decarboxylase (GAD) is the key enzyme to generate GABA. A simple and economical method of preparing and immobilizing GAD would be helpful for GABA production. In this study, the GAD from Lactobacillus fermentum YS2 was expressed under the control of a stress-inducible promoter and was purified and immobilized in a fusion form, and its reusability was investigated. Results: The fusion protein CBM-GAD was expressed in Escherichia coli DH5α carrying pCROCB-gadB, which contained promoter PrpoS, cbm3 (family 3 carbohydrate-binding module from Clostridium thermocellum) coding sequence, the gadB gene from L. fermentum YS2 coding for GAD, and the T7 terminator. After a one-step purification of CBM-GAD using regenerated amorphous cellulose (RAC) as an adsorbent, SDS-PAGE analysis revealed a clear band of 71 kDa; the specific activity of the purified fusion protein CBM-GAD reached 83.6 ± 0.7 U·mg-1. After adsorption onto RAC, the immobilized GAD with CBM3 tag was repeatedly used for GABA synthesis. The protein-binding capacity of RAC was 174 ± 8 mg·g-1. The immobilized CBM-GAD could repeatedly catalyze GABA synthesis, and 8% of the initial activities was retained after 10 uses. We tested the conversion of monosodium glutamate to GABA by the immobilized enzyme; the yield reached 5.15 g/L and the productivity reached 3.09 g/L·h. Conclusions: RAC could be used as an adsorbent in one-step purification and immobilization of CBM-GAD, and the immobilized enzyme could be repeatedly used to catalyze the conversion of glutamate to GABA.

Engineering and characterization of new intrinsic transcriptional terminators in Bacillus subtilis 168 / 生物工程学报

Terminators as regulatory signals are typically placed behind the last coding sequence to block the transcription of DNA to RNA and release the transcript. In the present study, the hairpin and the U-rich sequence of the bacteriophage λto terminator were first modified to investigate their effects on termination efficiency and mRNA stability in Bacillus subtilis 168. Compared with the native λto terminator, the terminator variants M3, M11 and M12 showed higher termination efficiency values. Moreover, the variantsM3, M4 and M11 showed significant positive effects on the mRNA stability of the upstream gfp gene. Additionally, insertion of RNase site also increased the mRNA stability. The results of this study suggested that the composition of the hairpin loop is not required for effective intrinsic termination in B. subtilis. Our results also showed that the terminator could also be used as a potential tool for increasing mRNA stability and the corresponding enzyme production in B. subtilis.

Genetic use restriction technologies and possible applications in the integrated pest management / Tecnologias genéticas de restrição de uso e possíveis aplicações no manejo integrado de pragas

ABSTRACT: Genetic use restriction technologies (GURTs) were developed to preserve the intellectual property of genetically modified crops (GM) and ensure the return of investments made by industry to obtain technology delivered through seeds. The aims of this review are to discuss the GURTs and analyze their possible applications in integrated management of agricultural pests. There are two classes of GURTs: T-GURTs (trait-based GURTs), wherein the generated seed are viable, but the next generation does not express the trait of agronomic interest, and V-GURT (variety-based GURTs), in which plants produce non viable seeds. However, beyond the seed protection purpose, the GURTs could have also other application to solve agronomic problems. One of the most important is the use of GURTs as a tool to restrict gene flow of GM traits to relative weeds. In addition, it is proposed the use of this technology in integrated weed management by preventing the GMs seed germination, which produces volunteer plants that compete with the crop of interest. Also, these volunteer plants may serve as alternative hosts for insects and pathogens in between crop seasons. The GURTs could contribute to the control of undesirable agents in agricultural systems, reducing the use of pesticides and increasing crop yields.

RESUMO: As tecnologias genéticas de restrição de uso (GURTs) foram idealizadas a fim de preservar a propriedade intelectual de culturas geneticamente modificadas (GMs) e garantir o retorno dos investimentos feitos pelas empresas para a obtenção de tecnologias transmitidas via sementes. Os objetivos dessa revisão são discutir as GURTs e analisar suas possíveis aplicações no manejo integrado de pragas agrícolas. Existem duas classes de GURTs: a T-GURT (trait-based GURT), na qual as sementes produzidas são viáveis, porém as plantas da geração seguinte não expressam o caractere de interesse agronômico, e a V-GURT (variety-based GURT), na qual as plantas produzem sementes inviáveis. Contudo, além do propósito da proteção das sementes, o uso de GURTs poderia também ter outras aplicações na resolução de problemas agronômicos. Um dos mais importantes é o uso das GURTs como ferramenta para impedir o fluxo de genes de culturas transgênicas para plantas daninhas coespecificas. Além disso, propõe-se o uso dessa tecnologia no manejo integrado de plantas daninhas, por meio da prevenção da germinação de sementes GMs, que geram plantas voluntárias que competem com a cultura de interesse. Além disso, essas plantas voluntárias podem servir como hospedeiros alternativos para insetos e patógenos nos períodos de entressafra. Dessa forma, as GURTs poderiam contribuir no controle de agentes indesejáveis em sistemas agrícolas, reduzindo a utilização de agrotóxicos e aumentando a produtividade dos cultivos.