Building a eukaryotic chromosome arm by de novo design and synthesis.

The genome of an organism is inherited from its ancestor and continues to evolve over time, however, the extent to which the current version could be altered remains unknown. To probe the genome plasticity of Saccharomyces cerevisiae, here we replace the native left arm of chromosome XII (chrXIIL) with a linear artificial chromosome harboring small sets of reconstructed genes. We find that as few as 12 genes are sufficient for cell viability, whereas 25 genes are required to recover the partial fitness defects observed in the 12-gene strain. Next, we demonstrate that these genes can be reconstructed individually using synthetic regulatory sequences and recoded open-reading frames with a "one-amino-acid-one-codon" strategy to remain functional. Finally, a synthetic neochromsome with the reconstructed genes is assembled which could substitute chrXIIL for viability. Together, our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional eukaryotic chromosomes from entirely artificial sequences.

A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis.

The coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously affected public health around the world. In-depth studies on the pathogenic mechanisms of SARS-CoV-2 is urgently necessary for pandemic prevention. However, most laboratory studies on SARS-CoV-2 have to be carried out in bio-safety level 3 (BSL-3) laboratories, greatly restricting the progress of relevant experiments. In this study, we used a bacterial artificial chromosome (BAC) method to assemble a SARS-CoV-2 replication and transcription system in Vero E6 cells without virion envelope formation, thus avoiding the risk of coronavirus exposure. Furthermore, an improved real-time quantitative reverse transcription PCR (RT-qPCR) approach was used to distinguish the replication of full-length replicon RNAs and transcription of subgenomic RNAs (sgRNAs). Using the SARS-CoV-2 replicon, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 facilitates the transcription of sgRNAs in the discontinuous synthesis process. Moreover, two high-frequency mutants of N protein, R203K and S194L, can obviously enhance the transcription level of the replicon, hinting that these mutations likely allow SARS-CoV-2 to spread and reproduce more quickly. In addition, remdesivir and chloroquine, two well-known drugs demonstrated to be effective against coronavirus in previous studies, also inhibited the transcription of our replicon, indicating the potential applications of this system in antiviral drug discovery. Overall, we developed a bio-safe and valuable replicon system of SARS-CoV-2 that is useful to study the mechanisms of viral RNA synthesis and has potential in novel antiviral drug screening.

Efficient de novo assembly and modification of large DNA fragments.

Synthetic genomics has provided new bottom-up platforms for the functional study of viral and microbial genomes. The construction of the large, gigabase (Gb)-sized genomes of higher organisms will deepen our understanding of genetic blueprints significantly. But for the synthesis and assembly of such large-scale genomes, the development of new or expanded methods is required. In this study, we develop an efficient pipeline for the construction of large DNA fragments sized 100 kilobases (kb) or above from scratches and describe an efficient method for "scar-free" engineering of the assembled sequences. Our method, therefore, should provide a standard framework for producing long DNA molecules, which are critical materials for synthetic genomics and metabolic engineering.

[Acupoint sensitization enhances inhibitory effect of electroacupuncture on jejunum mobility in rats].

OBJECTIVE: To observe the effect difference of electroacupuncture (EA) at the sensitized and non-sensitized acupoint "Tianshu" (ST25) on the motility of jejunum in rats. METHODS: Male SD rats were randomly divided into blank control, sensitized ST25 (sensitization), and non-sensitized ST25 (non-sensitization) groups (n=16 in each group). The sensitization and non-sensitization were induced by injection of 15% mustard oil (20 µL) and Paraffin oil (20 µL) into the left ST25 area respectively. The rats' behavior reactions were assessed by recording the numbers and duration of licking the local sensitized skin region. At the end of experiments and after regular trans-cardiac perfusion and fixing with 10% neutral formaldehyde, the skin and muscular tissues of the ST25 region (1 cm×1 cm) were taken for H.E. staining to observe the local histopathologic changes. The intestinal motility was detected by recording the contraction pressure waves of jejunum through a small balloon-connected pressure transducer and an amplifier, followed by calculating the ratios of amplitude and frequency of contraction waves between pre- and post-EA stimulation. EA (2 Hz, 0.2-6.0 mA) was applied to the sensitized and non-sensitized ST25 regions for 20 s for comparing their effects on jejunum motility. RESULTS: Compared with the blank control group, mustard oil injection but not Paraffin oil injection resulted in histological edema and neutrophils infiltration in tissues of ST25 region, and also striking increase of the number and duration of licking (P<0.01), suggesting a sensitization of ST25 area after mustard oil injection. The ratios of spontaneous contraction amplitude and frequency of jejunum were remarkably decreased in mustard oil-treated rats compared to baseline and Paraffin oil group (P<0.001，P<0.01). The maximum inhibitory effect of EA on the intestinal movement amplitude was significantly higher in the sensitization group than in the blank control and non-sensitization groups (P<0.05，P<0.01), and there was a dose-effect relationship between the current intensity of EA (0.5-3.0 mA) and the inhibitory rate 50% of the contraction amplitude (not the frequency) in the sensitization group (P<0.001), but not in the blank control and non-sensitization groups (P>0.05). Additionally, 1.5 mA EA stimulation at the sensitized ST25 (not at the non-sensitized ST25) had an inhibitory effect on the contraction amplitude (P<0.05) rather than on the contraction frequency (P>0.05) in comparison with the blank control group. CONCLUSION: Sensitization of acupoint ST25 can modulate the motility of jejunum and enhances the inhibitory effect of EA on the contraction amplitude of jejunum in rats. The inhibitory effect of sensitized ST25 EA is evidently stronger than that of the non-sensitized ST25 EA.

[Manual acupuncture stimulation of acupoints at the same and adjacent spinal segments promotes uterine contractility].

OBJECTIVE: To investigate the effect of manual acupuncture stimulation of acupoints at different spinal nerve segments on uterine motility and the complicated adrenergic and cholinergic receptors. METHODS: Eighteen adult non-pregnant SD rats were used in the present study. The contractive activities of the uterus were measured by using a pressure transducer which was connected to an inserted water-filled balloon in the uterus via a PE tube at one end and an amplifier at the other end. Manual acupuncture needle was applied to "Zigong"(EX-CA1),"Huiyin" (CV1), "Xuehai "(SP10) and "Taichong "(LR3) acupoints located at the same or adjacent spinal nerve segments of the uterus, and to "Neiguan" (PC6) situated at the distant spinal nerve segment at about 2 Hz for 1 min, followed by observing changes of the uterine contractility. After acupuncture, α-adrenoceptor antagonist phentolamine (0.5 mg/kg, n=9) or cholinergic muscarinic (M) receptor antagonist atropine (0.5 mg/kg，n=9) was given to the rats of different acupoints respectively through tail vein, followed by observing changes of the uterine automatic systolic pressure difference (value of systolic pressure peak minuses the trough value) and frequency after manual acupuncture stimulation as well as after blocking the activities of α-adrenoceptors and M receptors, separately. RESULTS: After acupuncture stimulation of EX-CA1, CV1, SP10 and LR3, but not PC6, the systolic pressure difference and frequency of the uterus were signi-ficantly increased (P<0.05,P<0.001, P<0.01). Following intravenous injection of phentolamine, both the systolic pressure difference and frequency had no marked changes after acupuncture at the 5 acupoints (P>0.05). After intravenous injection of atropine, the uterine systolic pressure difference and frequency were markedly decreased compared with the basic values before acupuncture stimulation (P<0.001), but had no obvious changes after acupuncture at the 5 acupoints at both the same and distant spinal segments to the uterus (P>0.05). CONCLUSION: Manual acupuncture stimulation of acupoints at the same and adjacent spinal segments can promote the contractility of uterus in normal rats, which is realized by activating both α-adrenoceptor and cholinergic M receptors.

Overproduction of L-tryptophan via simultaneous feed of glucose and anthranilic acid from recombinant Escherichia coli W3110: Kinetic modeling and process scale-up.

L-tryptophan is an essential amino acid widely used in food and pharmaceutical industries. However, its production via Escherichia coli fermentation suffers severely from both low glucose conversion efficiency and acetic acid inhibition, and to date effective process control methods have rarely been explored to facilitate its industrial scale production. To resolve these challenges, in the current research an engineered strain of E. coli was used to overproduce L-tryptophan. To achieve this, a novel dynamic control strategy which incorporates an optimized anthranilic acid feeding into a dissolved oxygen-stat (DO-stat) glucose feeding framework was proposed for the first time. Three original contributions were observed. Firstly, compared to previous DO control methods, the current strategy was able to inhibit completely the production of acetic acid, and its glucose to L-tryptophan yield reached 0.211 g/g, 62.3% higher than the previously reported. Secondly, a rigorous kinetic model was constructed to simulate the underlying biochemical process and identify the effect of anthranilic acid on both glucose conversion and L-tryptophan synthesis. Finally, a thorough investigation was conducted to testify the capability of both the kinetic model and the novel control strategy for process scale-up. It was found that the model possesses great predictive power, and the presented strategy achieved the highest glucose to L-tryptophan yield (0.224 g/g) ever reported in large scale processes, which approaches the theoretical maximum yield of 0.227 g/g. This research, therefore, paves the way to significantly enhance the profitability of the investigated bioprocess.