Identification of Bromophenols' glucuronidation and its induction on UDP- glucuronosyltransferases isoforms.

Bromophenols (BPs) are prominent environmental pollutants extensively utilized in aquaculture, pharmaceuticals, and chemical manufacturing. This study aims to identify UDP- glucuronosyltransferases (UGTs) isoforms involved in the metabolic elimination of BPs. Mono-glucuronides of BPs were detected in human liver microsomes (HLMs) incubated with the co-factor uridine-diphosphate glucuronic acid (UDPGA). The glucuronidation metabolism reactions catalyzed by HLMs followed Michaelis-Menten or substrate inhibition kinetics. Recombinant enzymes and inhibition experiments with chemical reagents were employed to phenotype the principal UGT isoforms participating in BP glucuronidation. UGT1A6 emerged as the major enzyme in the glucuronidation of 4-Bromophenol (4-BP), while UGT1A1, UGT1A6, and UGT1A8 were identified as the most essential isoforms for metabolizing 2,4-dibromophenol (2,4-DBP). UGT1A1, UGT1A8, and UGT2B4 were deemed the most critical isoforms in the catalysis of 2,4,6-tribromophenol (2,4,6-TBP) glucuronidation. Species differences were investigated using the liver microsomes of pig (PLM), rat (RLM), monkey (MyLM), and dog (DLM). Additionally, 2,4,6-TBP effects on the expression of UGT1A1 and UGT2B7 in HepG2 cells were evaluated. The results demonstrated potential induction of UGT1A1 and UGT2B7 upon exposure to 2,4,6-TBP at a concentration of 50 µM. Collectively, these findings contribute to elucidating the metabolic elimination and toxicity of BPs.

Assessment of the inhibition risk of chlorophenol substances on cytochrome P450 via cocktail inhibition assays.

Chlorophenols (CPs) are widespread pollutants in nature. CPs have raised significant concern due to their potential hepatotoxic effects on humans. This research aimed to ascertain the inhibitory potential of eleven CPs (2-CP, 3-CP, 4-CP, 2,4-DCP, 2,3,4-TCP, 2,4,5-TCP, 2,4,6-TCP, 2,3,4,5-TeCP, 2,3,4,6-TeCP, 2,3,5,6-TeCP, and PCP) on nine human CYP isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4). The CPs that inhibit the activity of CYP isoforms were detected with human liver microsomes (HLM) using a cocktail approach in vitro. The results demonstrated that trichlorophenols, tetrachlorophenols, and PCP strongly inhibited CYP2C8 and CYP2C9. The half inhibition concentration (IC50) value of 2,3,4,6-TeCP and PCP for CYP2C8 inhibition was 27.3 µM and 12.3 µM, respectively. The IC50 for the inhibition of 2,4,6-TCP, 2,3,4,6-TeCP and PCP towards CYP2C9 were calculated to be 30.3 µM, 5.8 µM and 2.2 µM, respectively. 2,3,4,6-TeCP, and PCP exhibited non-competitive inhibition towards CYP2C8. 2,4,6-TCP, 2,3,4,6-TeCP, and PCP exhibited competitive inhibition towards CYP2C9. The inhibition kinetics parameters (Ki) were 51.51 µM, 22.28 µM, 37.86 µM, 7.27 µM, 0.68 µM for 2,3,4,6-TeCP-CYP2C8, PCP-CYP2C8, 2,4,6-TCP-CYP2C9, 2,3,4,6-TeCP-CYP2C9, PCP-CYP2C9, respectively. This study also defined clear structure-activity relationships (SAR) of CPs on CYP2C8, supported by molecular docking studies. Overall, CPs were found to cause inhibitory effects on CYP isoforms in vitro, and this finding may provide a basis for CPs focused on CYP isoforms inhibition endpoints.

Engineering Pseudomonas entomophila for synthesis of copolymers with defined fractions of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates.

Polyhydroxyalkanoates (PHA) composed of both short-chain-length (SCL) and medium-chain-length (MCL) monomers (SCL-co-MCL PHA) combine the advantages of high strength and elasticity provided by SCL PHA and MCL PHA, respectively. Synthesis of SCL-co-MCL PHA, namely, copolymers of 3-hydroxybutyrate (3HB) and MCL 3-hydroxyalkanoates (3HA) such as 3-hydroxydecanoate (3HD) and longer chain 3HA, has been a challenge for a long time. This study aims to engineer Pseudomonas entomophila for synthesizing P(3HB-co-MCL 3HA) via weakening its ß-oxidation pathway combined with insertion of 3HB synthesis pathway consisting of ß-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB). 3HB and MCL 3HA polymerization is catalyzed by a low specificity PHA synthase (phaC), namely, mutated PhaC61-3. The link between the fatty acid de novo synthesis and PHA synthesis was further blocked to increase the supply for SCL and MCL monomers in P. entomophila. The so-constructed P. entomophila was successfully used to synthesize novel PHA copolymers of P(3HB-co-3HD), P(3HB-co-3HDD) and P(3HB-co-3H9D) consisting of 3HB and 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decanent (3H9D), respectively. MCL 3HA compositions of P(3HB-co-3HD) and P(3HB-co-3HDD) can be adjusted from 0 to approximate 100 mol%. Results demonstrated that the engineered P. entomophila could be a platform for tailor-made P(3HB-co-MCL 3HA).

Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] is one of the most promising biomaterials expected to be used in a wide range of scenarios. However, its large-scale production is still hindered by the high cost. Here we report the engineering of Halomonas bluephagenesis as a low-cost platform for non-sterile and continuous fermentative production of P(3HB-co-4HB) from glucose. Two interrelated 4-hydroxybutyrate (4HB) biosynthesis pathways were constructed to guarantee 4HB monomer supply for P(3HB-co-4HB) synthesis by working in concert with 3-hydroxybutyrate (3HB) pathway. Interestingly, only 0.17 mol% 4HB in the copolymer was obtained during shake flask studies. Pathway debugging using structurally related carbon source located the failure as insufficient 4HB accumulation. Further whole genome sequencing and comparative genomic analysis identified multiple orthologs of succinate semialdehyde dehydrogenase (gabD) that may compete with 4HB synthesis flux in H. bluephagenesis. Accordingly, combinatory gene-knockout strains were constructed and characterized, through which the molar fraction of 4HB was increased by 24-fold in shake flask studies. The best-performing strain was grown on glucose as the single carbon source for 60 h under non-sterile conditions in a 7-L bioreactor, reaching 26.3 g/L of dry cell mass containing 60.5% P(3HB-co-17.04 mol%4HB). Besides, 4HB molar fraction in the copolymer can be tuned from 13 mol% to 25 mol% by controlling the residual glucose concentration in the cultures. This is the first study to achieve the production of P(3HB-co-4HB) from only glucose using Halomonas.

Novel T7-like expression systems used for Halomonas.

To engineer non-model organisms, suitable genetic parts must be available. However, biological parts are often host strain sensitive. It is therefore necessary to develop genetic parts that are functional regardless of host strains. Here we report several novel phage-derived expression systems used for transcriptional control in non-model bacteria. Novel T7-like RNA polymerase-promoter pairs were obtained by mining phage genomes, followed by in vivo characterization in non-model strains Halomonas spp TD01 and Pseudomonas entomophila. Three expression systems, namely, MmP1, VP4, and K1F, were developed displaying orthogonality (crosstalk<0.7%), tight regulation (3085-fold induction), and high efficiency (2.5-fold of Ptac) in Halomonas sp. TD01, a chassis strain with a high industrial value. The expression under the corresponding T7-like promoter libraries persisted with striking correlations (R2 >0.94) between Escherichia coli and Halomonas sp. TD01, implying suitability of broad-host range. Three Halomonas TD strains were then constructed based upon these expression systems that enabled interchangeable and controllable gene expression. One of the strains termed Halomonas TD-MmP1 was used to express the cell-elongation cassette (minCD genes) and polyhydroxybutyrate (PHB) biosynthetic pathway, resulting in a 100-fold increase in cell lengths and high levels of PHB production (up to 92% of cell dry weight), respectively. We envision these T7-like expression systems to benefit metabolic engineering in other non-model organisms.

Elevated MMP-9 in the lumbar cord early after thoracic spinal cord injury impedes motor relearning in mice.

Spinal cord injury results in distant pathology around putative locomotor networks that may jeopardize the recovery of locomotion. We previously showed that activated microglia and increased cytokine expression extend at least 10 segments below the injury to influence sensory function. Matrix metalloproteinase-9 (MMP-9) is a potent regulator of acute neuroinflammation. Whether MMP-9 is produced remote to the injury or influences locomotor plasticity remains unexamined. Therefore, we characterized the lumbar enlargement after a T9 spinal cord injury in C57BL/6 (wild-type [WT]) and MMP-9-null (knock-out [KO]) mice. Within 24 h, resident microglia displayed an activated phenotype alongside increased expression of progelatinase MMP-3 in WT mice. By 7 d, increases in active MMP-9 around lumbar vasculature and production of proinflammatory TNF-α were evident. Deletion of MMP-9 attenuated remote microglial activation and restored TNF-α expression to homeostatic levels. To determine whether MMP-9 impedes locomotor plasticity, we delivered lumbar-focused treadmill training in WT and KO mice during early (2-9 d) or late (35-42 d) phases of recovery. Robust behavioral improvements were observed by 7 d, when only trained KO mice stepped in the open field. Locomotor improvements were retained for 4 weeks as identified using state of the art mouse kinematics. Neither training nor MMP-9 depletion alone promoted recovery. The same intervention delivered late was ineffective, suggesting that lesion site sparing is insufficient to facilitate activity-based training and recovery. Our work suggests that by attenuating remote mechanisms of inflammation, acute treadmill training can harness endogenous spinal plasticity to promote robust recovery.

A bilayer bioengineered patch with sequential dual-growth factor release to promote vascularization in bladder reconstruction.

Bladder tissue engineering holds promise for addressing bladder defects resulting from congenital or acquired bladder diseases. However, inadequate vascularization significantly impacts the survival and function of engineered tissues after transplantation. Herein, a novel bilayer silk fibroin (BSF) scaffold was fabricated with the capability of vascular endothelial growth factor (VEGF) and platelet derived growth factor-BB (PDGF-BB) sequential release. The outer layer of the scaffold was composed of compact SF film with waterproofness to mimic the serosa of the bladder. The inner layer was constructed of porous SF matrix incorporated with SF microspheres (MS) loaded with VEGF and PDGF-BB. We found that the 5% (w/v) MS-incorporated scaffold exhibited a rapid release of VEGF, whereas the 0.2% (w/v) MS-incorporated scaffold demonstrated a slow and sustained release of PDGF-BB. The BSF scaffold exhibited good biocompatibility and promoted endothelial cell migration, tube formation and enhanced endothelial differentiation of adipose derived stem cells (ADSCs) in vitro. The BSF patch was constructed by seeding ADSCs on the BSF scaffold. After in vivo transplantation, not only could the BSF patch facilitate the regeneration of urothelium and smooth muscle, but more importantly, stimulate the regeneration of blood vessels. This study demonstrated that the BSF patch exhibited excellent vascularization capability in bladder reconstruction and offered a viable functional bioengineered patch for future clinical studies.

Precise programming of multigene expression stoichiometry in mammalian cells by a modular and programmable transcriptional system.

Context-dependency of mammalian transcriptional elements has hindered the quantitative investigation of multigene expression stoichiometry and its biological functions. Here, we describe a host- and local DNA context-independent transcription system to gradually fine-tune single and multiple gene expression with predictable stoichiometries. The mammalian transcription system is composed of a library of modular and programmable promoters from bacteriophage and its cognate RNA polymerase (RNAP) fused to a capping enzyme. The relative expression of single genes is quantitatively determined by the relative binding affinity of the RNAP to the promoters, while multigene expression stoichiometry is predicted by a simple biochemical model with resource competition. We use these programmable and modular promoters to predictably tune the expression of three components of an influenza A virus-like particle (VLP). Optimized stoichiometry leads to a 2-fold yield of intact VLP complexes. The host-independent orthogonal transcription system provides a platform for dose-dependent control of multiple protein expression which may be applied for advanced vaccine engineering, cell-fate programming and other therapeutic applications.

Matrix metalloproteinase-9 and stromal cell-derived factor-1 act synergistically to support migration of blood-borne monocytes into the injured spinal cord.

The infiltration of monocytes into the lesioned site is a key event in the inflammatory response after spinal cord injury (SCI). We hypothesized that the molecular events governing the infiltration of monocytes into the injured cord involve cooperativity between the upregulation of the chemoattractant stromal cell-derived factor-1 (SDF-1)/CXCL12 in the injured cord and matrix metalloproteinase-9 (MMP-9/gelatinase B), expressed by infiltrating monocytes. SDF-1 and its receptor CXCR4 mRNAs were upregulated in the injured cord, while macrophages immunoexpressed CXCR4. When mice, transplanted with bone marrow cells from green fluorescent protein (GFP) transgenic mice, were subjected to SCI, GFP+ monocytes infiltrated the cord and displayed gelatinolytic activity. In vitro studies confirmed that SDF-1α, acting through CXCR4, expressed on bone marrow-derived macrophages, upregulated MMP-9 and stimulated MMP-9-dependent transmigration across endothelial cell monolayers by 2.6-fold. There was a reduction in F4/80+ macrophages in spinal cord-injured MMP-9 knock-out mice (by 36%) or wild-type mice, treated with the broad-spectrum MMP inhibitor GM6001 (by 30%). Mice were adoptively transferred with myeloid cells and treated with the MMP-9/-2 inhibitor SB-3CT, the CXCR4 antagonist AMD3100, or a combination of both drugs. While either drug resulted in a 28-30% reduction of infiltrated myeloid cells, the combined treatment resulted in a 45% reduction, suggesting that SDF-1 and MMP-9 function independently to promote the trafficking of myeloid cells into the injured cord. Collectively, these observations suggest a synergistic partnership between MMP-9 and SDF-1 in facilitating transmigration of monocytes into the injured spinal cord.

Keratan sulfate restricts neural plasticity after spinal cord injury.

Chondroitin sulfate (CS) proteoglycans are strong inhibitors of structural rearrangement after injuries of the adult CNS. In addition to CS chains, keratan sulfate (KS) chains are also covalently attached to some proteoglycans. CS and KS sometimes share the same core protein, but exist as independent sugar chains. However, the biological significance of KS remains elusive. Here, we addressed the question of whether KS is involved in plasticity after spinal cord injury. Keratanase II (K-II) specifically degraded KS, i.e., not CS, in vivo. This enzyme digestion promoted the recovery of motor and sensory function after spinal cord injury in rats. Consistent with this, axonal regeneration/sprouting was enhanced in K-II-treated rats. K-II and the CS-degrading enzyme chondroitinase ABC exerted comparable effects in vivo and in vitro. However, these two enzymes worked neither additively nor synergistically. These data and further in vitro studies involving artificial proteoglycans (KS/CS-albumin) and heat-denatured or reduced/alkylated proteoglycans suggested that all three components of the proteoglycan moiety, i.e., the core protein, CS chains, and KS chains, were required for the inhibitory activity of proteoglycans. We conclude that KS is essential for, and has an impact comparable to that of CS on, postinjury plasticity. Our study also established that KS and CS are independent requirements for the proteoglycan-mediated inhibition of axonal regeneration/sprouting.

N-acetylglucosamine 6-O-sulfotransferase-1-deficient mice show better functional recovery after spinal cord injury.

Neurons in the adult CNS do not spontaneously regenerate after injuries. The glycosaminoglycan keratan sulfate is induced after spinal cord injury, but its biological significance is not well understood. Here we investigated the role of keratan sulfate in functional recovery after spinal cord injury, using mice deficient in N-acetylglucosamine 6-O-sulfotransferase-1 that lack 5D4-reactive keratan sulfate in the CNS. We made contusion injuries at the 10th thoracic level. Expressions of N-acetylglucosamine 6-O-sulfotransferase-1 and keratan sulfate were induced after injury in wild-type mice, but not in the deficient mice. The wild-type and deficient mice showed similar degrees of chondroitin sulfate induction and of CD11b-positive inflammatory cell recruitment. However, motor function recovery, as assessed by the footfall test, footprint test, and Basso mouse scale locomotor scoring, was significantly better in the deficient mice. Moreover, the deficient mice showed a restoration of neuromuscular system function below the lesion after electrical stimulation at the occipito-cervical area. In addition, axonal regrowth of both the corticospinal and raphespinal tracts was promoted in the deficient mice. In vitro assays using primary cerebellar granule neurons demonstrated that keratan sulfate proteoglycans were required for the proteoglycan-mediated inhibition of neurite outgrowth. These data collectively indicate that keratan sulfate expression is closely associated with functional disturbance after spinal cord injury. N-acetylglucosamine 6-O-sulfotransferase-1-deficient mice are a good model to investigate the roles of keratan sulfate in the CNS.

Feedback activation of STAT3 limits the response to PI3K/AKT/mTOR inhibitors in PTEN-deficient cancer cells.

The PI3K/AKT/mTOR signaling pathway is constitutively active in PTEN-deficient cancer cells, and its targeted inhibition has significant anti-tumor effects. However, the efficacy of targeted therapies is often limited due to drug resistance. The relevant signaling pathways in PTEN-deficient cancer cells treated with the PI3K/mTOR inhibitor BEZ235 were screened using a phosphokinase array, and further validated following treatment with multiple PI3K/AKT/mTOR inhibitors or AKT knockdown. The correlation between PTEN expression levels and STAT3 kinase phosphorylation in the tissue microarrays of gastric cancer patients was analyzed by immunohistochemistry. Cell proliferation and clonogenic assays were performed on the suitably treated PTEN-deficient cancer cells. Cytokine arrays, small molecule inhibition and knockdown assays were performed to identify related factors. PTEN-deficient tumor xenografts were established in nude mice that were treated with PI3K/AKT/mTOR and/or STAT3 inhibitors. PTEN deficiency was positively correlated with low STAT3 activity. PI3K/mTOR inhibitors increased the expression and secretion of macrophage migration inhibitory factor (MIF) and activated the JAK1/STAT3 signaling pathway. Both cancer cells and in vivo tumor xenografts showed that the combined inhibition of PI3K/AKT/mTOR and STAT3 activity enhanced the inhibitory effect of BEZ235 on the proliferation of PTEN-deficient cancer cells. Our findings provide a scientific basis for a novel treatment strategy in cancer patients with PTEN deficiency.

Cryoprecipitate attenuates the endotheliopathy of trauma in mice subjected to hemorrhagic shock and trauma.

BACKGROUND: Plasma has been shown to mitigate the endotheliopathy of trauma. Protection of the endothelium may be due in part to fibrinogen and other plasma-derived proteins found in cryoprecipitate; however, the exact mechanisms remain unknown. Clinical trials are underway investigating early cryoprecipitate administration in trauma. In this study, we hypothesize that cryoprecipitate will inhibit endothelial cell (EC) permeability in vitro and will replicate the ability of plasma to attenuate pulmonary vascular permeability and inflammation induced by hemorrhagic shock and trauma (HS/T) in mice. METHODS: In vitro, barrier permeability of ECs subjected to thrombin challenge was measured by transendothelial electrical resistance. In vivo, using an established mouse model of HS/T, we compared pulmonary vascular permeability among mice resuscitated with (1) lactated Ringer's solution (LR), (2) fresh frozen plasma (FFP), or (3) cryoprecipitate. Lung tissue from the mice in all groups was analyzed for markers of vascular integrity, inflammation, and inflammatory gene expression via NanoString messenger RNA quantification. RESULTS: Cryoprecipitate attenuates EC permeability and EC junctional compromise induced by thrombin in vitro in a dose-dependent fashion. In vivo, resuscitation of HS/T mice with either FFP or cryoprecipitate attenuates pulmonary vascular permeability (sham, 297 ± 155; LR, 848 ± 331; FFP, 379 ± 275; cryoprecipitate, 405 ± 207; p < 0.01, sham vs. LR; p < 0.01, LR vs. FFP; and p < 0.05, LR vs. cryoprecipitate). Lungs from cryoprecipitate- and FFP-treated mice demonstrate decreased lung injury, decreased infiltration of neutrophils and activation of macrophages, and preserved pericyte-endothelial interaction compared with LR-treated mice. Gene analysis of lung tissue from cryoprecipitate- and FFP-treated mice demonstrates decreased inflammatory gene expression, in particular, IL-1ß and NLRP3, compared with LR-treated mice. CONCLUSION: Our data suggest that cryoprecipitate attenuates the endotheliopathy of trauma in HS/T similar to FFP. Further investigation is warranted on active components and their mechanisms of action.

De novo design of an intercellular signaling toolbox for multi-channel cell-cell communication and biological computation.

Intercellular signaling is indispensable for single cells to form complex biological structures, such as biofilms, tissues and organs. The genetic tools available for engineering intercellular signaling, however, are quite limited. Here we exploit the chemical diversity of biological small molecules to de novo design a genetic toolbox for high-performance, multi-channel cell-cell communications and biological computations. By biosynthetic pathway design for signal molecules, rational engineering of sensing promoters and directed evolution of sensing transcription factors, we obtain six cell-cell signaling channels in bacteria with orthogonality far exceeding the conventional quorum sensing systems and successfully transfer some of them into yeast and human cells. For demonstration, they are applied in cell consortia to generate bacterial colony-patterns using up to four signaling channels simultaneously and to implement distributed bio-computation containing seven different strains as basic units. This intercellular signaling toolbox paves the way for engineering complex multicellularity including artificial ecosystems and smart tissues.

Matrix metalloproteinase-9 facilitates glial scar formation in the injured spinal cord.

In the injured spinal cord, a glial scar forms and becomes a major obstacle to axonal regeneration. Formation of the glial scar involves migration of astrocytes toward the lesion. Matrix metalloproteinases (MMPs), including MMP-9 and MMP-2, govern cell migration through their ability to degrade constituents of the extracellular matrix. Although MMP-9 is expressed in reactive astrocytes, its involvement in astrocyte migration and formation of a glial scar is unknown. Here we found that spinal cord injured, wild-type mice expressing MMPs developed a more severe glial scar and enhanced expression of chondroitin sulfate proteoglycans, indicative of a more inhibitory environment for axonal regeneration/plasticity, than MMP-9 null mice. To determine whether MMP-9 mediates astrocyte migration, we conducted a scratch wound assay using astrocytes cultured from MMP-9 null, MMP-2 null, and wild-type mice. Gelatin zymography confirmed the expression of MMP-9 and MMP-2 in wild-type cultures. MMP-9 null astrocytes and wild-type astrocytes, treated with an MMP-9 inhibitor, exhibited impaired migration relative to untreated wild-type controls. MMP-9 null astrocytes showed abnormalities in the actin cytoskeletal organization and function but no detectable untoward effects on proliferation, cellular viability, or adhesion. Interestingly, MMP-2 null astrocytes showed increased migration, which could be attenuated in the presence of an MMP-9 inhibitor. Collectively, our studies provide explicit evidence that MMP-9 is integral to the formation of an inhibitory glial scar and cytoskeleton-mediated astrocyte migration. MMP-9 may thus be a promising therapeutic target to reduce glial scarring during wound healing after spinal cord injury.

[Preface for special column on iGEM (2018)].

Themed in synthetic biology, supplemented with multiple inter-disciplines, International Genetically Engineered Machine (iGEM)Competition provides with a most influential and dynamic platform for young minds in the field of biology. Many college and high school teams not only achieved excellent results in this competition, but got academic breakthrough achievements as well in recent years. Hence, we launch this iGEM special editorial column, featured in latest domestic iGEM research projects. Simultaneously, we discuss, in this special issue, about the progress of iGEM in China, and its inspiration on development of research skills and scientific competence of collegiate students.

[Bringing scientific research education closer to undergraduates through International Genetically Engineered Machine competition].

In recent years, the International Genetically Engineered Machine (iGEM) competition has experienced rapid global development. In 2017 alone, the number of iGEM teams registered around the globe reached an unprecedented 313, with 98 iGEM teams from China having enrolled in the competition and obtained outstanding results. In contrast to the many college students' innovation projects and scientific research training programs in China, iGEM's organization mode is focused on student-centered research learning. Moreover, it achieved a rich educational effect, embodying a new educational idea, which gives it great significance for the extracurricular scientific research training of undergraduates in Chinese universities. In this article, we took Peking University's participation in the iGEM competition as a starting point. The first part introduces the background and general situation of the iGEM competition. The second part reproduces the general procedure of one iGEM season and organization of Peking University's team. The third part compares iGEM's organization mode with those of other undergraduate research training courses and discusses them in detail. The fourth part sums up the experience with iGEM activities as well as explains its effect on developing the research capacity of undergraduate students as well as inspiring them to organize an undergraduate scientific research competition. This article aims to provide a reference for the organization of iGEM activities in domestic universities and for the reform of undergraduate education.

Engineering of a genetic circuit with regulatable multistability.

Synthetic biologists are dedicated to designing genetic systems from the bottom up to understand how living systems work. To date, a variety of genetic circuits exhibiting bistability have been designed, greatly expanding our understanding of the biological multistability in natural systems. However, the study of more complex forms of biological multistability using synthetic methods is still limited. In this report, we describe the engineering of a genetic circuit with regulatable multistability. A novel genetic toggle switch exhibiting inducible bistability and a self-activation circuit were individually designed and characterized, after which they were assembled to create a circuit that presents tristability. In bacteria, this synthetic circuit enables cells to differentiate spontaneously into three different states of gene expression. Moreover, the multistability of the circuit can be modulated by external inputs. This work provides a synthetic biology framework for the study of biological multistability and may help to understand natural multistability phenomena.

Pilot Scale-up of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Production by Halomonas bluephagenesis via Cell Growth Adapted Optimization Process.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB), is one of the most valuable biopolymers because of its flexible mechanical properties. In this study, the goal is to establish a scaled-up process of low cost P(3HB-co-4HB) from a 7.5-L fermentor to 1- and 5-m3 industrial bioreactors, respectively, using Halomonas bluephagenesis TD40 grown on glucose, γ-butyrolactone, and waste corn steep liquor (CSL) as substrates, under open non-sterile and fed-batch or continuous conditions. The non-sterile process enables the energy reduction for less steam consumption. Moreover, waste gluconate is successfully utilized to replace glucose as a carbon source for cell growth and PHA accumulation in 7.5-L fermentor, which opens the possibility of 60% of raw material cost reduction for recycling the waste resources. A mathematical model and rational calculation is established to help guide the feeding strategy and scale-up, respectively, leading to 100 g L-1 cell dry weight (CDW) containing 60.4% P(3HB-co-mol 13.5% 4HB) after 36 h of growth in the 5 m3 vessel. An even higher P(3HB-co-4HB) content of 74% is achieved by decreasing the use of waste CSL. A stable and continuous open process for efficient low-cost production of P(3HB-co-4HB) is successfully developed coupling fermentation with the downstream extraction processing.

Insulated transcriptional elements enable precise design of genetic circuits.

Rational engineering of biological systems is often complicated by the complex but unwanted interactions between cellular components at multiple levels. Here we address this issue at the level of prokaryotic transcription by insulating minimal promoters and operators to prevent their interaction and enable the biophysical modeling of synthetic transcription without free parameters. This approach allows genetic circuit design with extraordinary precision and diversity, and consequently simplifies the design-build-test-learn cycle of circuit engineering to a mix-and-match workflow. As a demonstration, combinatorial promoters encoding NOT-gate functions were designed from scratch with mean errors of <1.5-fold and a success rate of >96% using our insulated transcription elements. Furthermore, four-node transcriptional networks with incoherent feed-forward loops that execute stripe-forming functions were obtained without any trial-and-error work. This insulation-based engineering strategy improves the resolution of genetic circuit technology and provides a simple approach for designing genetic circuits for systems and synthetic biology.Unwanted interactions between cellular components can complicate rational engineering of biological systems. Here the authors design insulated minimal promoters and operators that enable biophysical modeling of bacterial transcription without free parameters for precise circuit design.