scholarly journals Deconstructing cell-free extract preparation forin vitroactivation of transcriptional genetic circuitry

2018 ◽  
Author(s):  
Adam D. Silverman ◽  
Nancy Kelley-Loughnane ◽  
Julius B. Lucks ◽  
Michael C. Jewett
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Genetic Circuits ◽  
Translation Rate ◽  
Bacterial Transcription ◽  
Crude Extracts ◽  
Run Off ◽  
Free Gene ◽  
Transcriptional Machinery

AbstractRecent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits designed as biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribedin vitroby the nativeEscherichia coliRNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial σ70promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture, but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing and will encourage the further proliferation of cell-free gene expression technology for new applications.

scholarly journals Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

2020 ◽  
Author(s):  
Antje Krüger ◽  
Alexander P. Mueller ◽  
Grant A. Rybnicky ◽  
Nancy L. Engle ◽  
Zamin K. Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Low Cost ◽  
Regulatory Elements ◽  
Free System ◽  
Gas Fermentation ◽  
Clostridium Autoethanogenum ◽  
Gene Regulatory Elements ◽  
Free Gene

ABSTRACTGas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic parts, gene expression, and biosynthetic pathway performance in vitro before implementing them in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 ± 24 µg/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

scholarly journals Borrelia burgdorferi Alters Its Gene Expression and Antigenic Profile in Response to CO2 Levels

2006 ◽  
Vol 189 (2) ◽  
pp. 437-445 ◽  
Author(s):  
Jenny A. Hyde ◽  
Jerome P. Trzeciakowski ◽  
Jonathan T. Skare
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Borrelia Burgdorferi ◽  
Growth Conditions ◽  
Etiologic Agent ◽  
Complex Life Cycle ◽  
Mammalian Host ◽  
Transcriptional Level ◽  
Virulence Determinants

ABSTRACT The etiologic agent of Lyme disease, Borrelia burgdorferi, must adapt to the distinct environments of its arthropod vector and mammalian host during its complex life cycle. B. burgdorferi alters gene expression and protein synthesis in response to temperature, pH, and other uncharacterized environmental factors. The hypothesis tested in this study is that dissolved gases, including CO2, serve as a signal for B. burgdorferi to alter protein production and gene expression. In this study we focused on characterization of in vitro anaerobic (5% CO2, 3% H2, 0.087 ppm O2) and microaerophilic (1% CO2, 3.48 ppm O2) growth conditions and how they modulate protein synthesis and gene expression in B. burgdorferi. Higher levels of several immunoreactive proteins, including BosR, NapA, DbpA, OspC, BBK32, and RpoS, were synthesized under anaerobic conditions. Previous studies demonstrated that lower levels of NapA were produced when microaerophilic cultures were purged with nitrogen gas to displace oxygen and CO2. In this study we identified CO2 as a factor contributing to the observed change in NapA synthesis. Specifically, a reduction in the level of dissolved CO2, independent of O2 levels, resulted in reduced NapA synthesis. BosR, DbpA, OspC, and RpoS synthesis was also decreased with the displacement of CO2. Quantitative reverse transcription-PCR indicated that the levels of the dbpA, ospC, and BBK32 transcripts are increased in the presence of CO2, indicating that these putative borrelial virulence determinants are regulated at the transcriptional level. Thus, dissolved CO2 may be an additional cue for borrelial host adaptation and gene regulation.

In-vitro culture of tobacco pollen: gene expression and protein synthesis

1992 ◽  
Vol 5 (4) ◽  
pp. 304-309 ◽  
Author(s):  
M. M. A. Van Herpen ◽  
P. F. M. de Groot ◽  
J. A. M. Schrauwen ◽  
K. J. P. T. van den Heuvel ◽  
K. A. P. Weterings ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
In Vitro Culture ◽  
Tobacco Pollen

scholarly journals Quantitative Modeling of Protein Synthesis Using Ribosome Profiling Data

2021 ◽  
Vol 8 ◽  
Author(s):  
Vandana Yadav ◽  
Inayat Ullah Irshad ◽  
Hemant Kumar ◽  
Ajeet K. Sharma
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Translation Initiation ◽  
Regulation Of Gene Expression ◽  
Ribosome Profiling ◽  
Quantitative Modeling ◽  
Quantitative Prediction ◽  
Translation Rate ◽  
The Past

Quantitative prediction on protein synthesis requires accurate translation initiation and codon translation rates. Ribosome profiling data, which provide steady-state distribution of relative ribosome occupancies along a transcript, can be used to extract these rate parameters. Various methods have been developed in the past few years to measure translation-initiation and codon translation rates from ribosome profiling data. In the review, we provide a detailed analysis of the key methods employed to extract the translation rate parameters from ribosome profiling data. We further discuss how these approaches were used to decipher the role of various structural and sequence-based features of mRNA molecules in the regulation of gene expression. The utilization of these accurate rate parameters in computational modeling of protein synthesis may provide new insights into the kinetic control of the process of gene expression.

scholarly journals Reconstruction of phospholipid synthesis by combing in vitro fatty acid synthesis and cell-free gene expression

2021 ◽  
Author(s):  
Sumie Eto ◽  
Rumie Matsumura ◽  
Mai Fujimi ◽  
Yasuhiro Shimane ◽  
Samuel Berhanu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Phospholipid Synthesis ◽  
Giant Vesicles ◽  
Phosphatidic Acids ◽  
A Cell ◽  
Free Gene

Phospholipid synthesis is a fundamental process that promotes cell propagation and, presently, is the most challenging issue in artificial cell research aimed at reconstituting living cells from biomolecules. Here, we constructed a cell-free phospholipid synthesis system that combines in vitro fatty acid synthesis and a cell-free gene expression system that synthesizes acyltransferases for phospholipid synthesis. Fatty acids were synthesized from acetyl-CoA and malonyl-CoA, then continuously converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of synthetic intermediates that suppress the reaction, the yield of phospholipid has significantly improved from previous schemes (up to 400 μM). Additionally, by adding enzymes for recycling CoA, we synthesized phosphatidic acids from acetic acid and bicarbonate as carbon sources. The constructed system is available to express the genes from pathogenic bacteria and to analyze the synthesized phospholipids. By encapsulating our system inside giant vesicles, it would be possible to construct the artificial cells in which the membrane grows and divides sustainably.

scholarly journals Induced Expression of the Legionella pneumophila Gene Encoding a 20-Kilodalton Protein during Intracellular Infection

1998 ◽  
Vol 66 (1) ◽  
pp. 203-212 ◽  
Author(s):  
Yousef Abu Kwaik
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Host Cell ◽  
Legionella Pneumophila ◽  
Cell Protein ◽  
Intracellular Bacteria ◽  
Host Cell Protein ◽  
Bacterial Gene ◽  
Intracellular Infection

ABSTRACT The eukaryotic protein synthesis inhibitor cycloheximid has been used by many investigators to selectively radiolabel intracellular bacteria. Although cycloheximide has no direct effect on bacterial gene expression, there are concerns that long-term inhibition of the host cell protein synthesis may have secondary effects on bacterial gene expression. Therefore, prior to further identification and cloning of the macrophage-induced (MI) genes of Legionella pneumophila, the effects of cycloheximide on L. pneumophila-infected U937 cells were evaluated by transmission electron microscopy. Inhibition of protein synthesis of the host cell for 6 h had no major effect on the ultrastructure of the host cell, on the formation of rough endoplasmic reticulum-surrounded replicative phagosome, or on initiation of intracellular bacterial replication. In contrast, by 15 h of cycloheximide treatment, there was profound deterioration in the host cell as well as in the phagosome. To examine protein synthesis by L. pneumophila during the intracellular infection, U937 macrophage-like cells were infected with L. pneumophila, and intracellular bacteria were radiolabeled during a 2-h cycloheximide treatment or following 12 h of cycloheximide treatment. Comparison by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein profile of radiolabeled in vitro-grown L. pneumophila to that of intracellularly radiolabeled bacteria showed that 23 proteins were induced in response to the intracellular environment during 2 h of inhibition of host cell protein biosynthesis. Twelve MI proteins ofL. pneumophila were artifactually induced due to prolonged inhibition of the host cell protein synthesis. The gene encoding a 20-kDa MI protein was cloned by a reverse genetics technique. Sequence analysis showed that the cloned gene encoded a protein that was 80% similar to the enzyme inorganic pyrophosphatase. Studies of promoter fusion to a promoterless lacZ gene showed that compared to in vitro-grown bacteria, expression of the pyrophosphatase gene (ppa) was induced fourfold throughout the intracellular infection. There was no detectable induction in transcription of the ppa promoter during exposure to stress stimuli in vitro. The ppa gene of L. pneumophila is the first example of a regulated ppagene which is selectively induced during intracellular infection and which may reflect enhanced capabilities of macromolecular biosynthesis by intracellular L. pneumophila. The data indicate caution in the long-term use of inhibition of host cell protein synthesis to selectively examine gene expression by intracellular bacteria.

scholarly journals AB0057 IN VITRO EFFECT OF CTLA4-IGG ON M1-M2 SHIFT OF MACROPHAGES FROM RHEUMATOID ARTHRITIS PATIENTS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1060.3-1061
Author(s):  
S. Tardito ◽  
S. Soldano ◽  
E. Gotelli ◽  
P. Montagna ◽  
S. Paolino ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Protein Synthesis ◽  
M2 Macrophage ◽  
Research Support ◽  
M1 Phenotype ◽  
Anti Inflammatory ◽  
Vitro Effect ◽  
M2 Phenotype

Background:Among the cells involved in the inflammatory process of rheumatoid arthritis (RA) [1], macrophages play a key role through their capacity to polarize into “classically” or “alternatively” activated phenotypes (M1 or M2) and making macrophages important players for the inflammatory cascade or for the anti-inflammatory reaction, respectively [2]. CTLA4-Ig fusion protein (abatacept) has been shown to contribute to macrophage shift from M1 to M2 [3].Objectives:We aimed to investigate the effects of abatacept to induce the polarization from the pro-inflammatory M1 phenotype into the anti-inflammatory M2 phenotype in cultured human macrophages obtained from RA patients’ and healthy subjects’(HS) circulating monocytes.Methods:Cultured monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of three early RA patients and ten HS, after signing informed consent and Ethics Committee approval. Cells were treated with phorbol myristate acetate (PMA) [5ng/ml] for 24 hours (hrs) to induce their differentiation into monocyte-derived macrophages (MDMs). Therefore, cultured HS MDMs were stimulated with lipopolysaccharides [LPS, 1mg/mL] for 4hrs [4] in order to induce their polarization into a pro-inflammatory M1 phenotype and then treated or not with abatacept at the concentrations of 100mg/mL and 500mg/mL for 3, 12, 24 and 48hrs. Cultured RA MDMs, were directly treated with abatacept as previous described. Cultured HS and RA MDMs without any pro-inflammatory stimuli and abatacept treatment were used as respective control.The transition of MDMs from M1 to M2 phenotype was evaluated through gene expression and protein synthesis of M2 macrophage markers, namely scavenger receptors (CD163 and CD204), and mannose receptor-1 (CD206) by quantitative real-time polymerase chain reaction (PCR) and by Western blotting. The statistical analysis evaluation was carried out by GraphPad Prism 8 analysis software using the Wilcoxon non-parametric t-test. Any p-value lower than 0.05 was considered as statistically significant. Results were indicated as median±standard deviation (SD).Results:In cultured RA MDMs (three cases), abatacept upregulated the gene expression of all investigated M2 markers, specifically after 12hrs of treatment with the concentration of 100mg/mL. In these cells, abatacept upregulated only the CD204 protein synthesis with more evidence at 24hrs of treatment and with the 500mg/mL concentration. In cultured HS MDMs (ten cases), abatacept upregulated the gene expression of M2 markers, significantly for that of CD206 [at 3hrs with 100mg/mL concentration, p= 0.0312] and CD163 [at 12hrs with 500mg/mL concentration, p= 0.0312]. Moreover, in these cells, abatacept significantly upregulated the protein synthesis of CD206 [at 48hrs with 500mg/mL concentration, p= 0.0195] and CD204 [at 24hrs with 100mg/mL concentration, p= 0.0156; both at 24 and 48hrs with 500mg/mL concentration, p= 0.0234].Conclusion:Preliminary data seem to indicate that abatacept can promote the in vitro shift from the M1 into the M2 macrophage phenotype, by upregulating specific markers (CD163, CD204, CD206) in cultured M1-MDMs from RA patients and in M1 macrophages induced from HS.References:[1]McInnes IB, et al. N Engl J Med 2011;365:2205–19.[2]Fujii M, et al. Biochem Biophys Res Commun. 2013;438(1):103-9.[3]Cutolo M, et al. Arthritis Res Ther. 2009;11:R176.[4]Pelegrin P., Surprenant, A. EMBO J. 2009 Jul 22; 28(14): 2114–2127.Disclosure of Interests:Samuele Tardito: None declared, Stefano Soldano: None declared, Emanuele Gotelli: None declared, Paola Montagna: None declared, Sabrina Paolino: None declared, Vanessa Smith: None declared, Maurizio Cutolo Grant/research support from: I received grant/research support from Bristol-Myers Squibb, Boehringer, Celgene.

scholarly journals CTLA4-Ig treatment induces M1–M2 shift in cultured monocyte-derived macrophages from healthy subjects and rheumatoid arthritis patients

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Maurizio Cutolo ◽  
Stefano Soldano ◽  
Emanuele Gotelli ◽  
Paola Montagna ◽  
Rosanna Campitiello ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Gene Expression ◽  
Protein Synthesis ◽  
Healthy Subjects ◽  
Mononuclear Cells ◽  
Costimulatory Molecule ◽  
High Concentration ◽  
Peripheral Blood Mononuclear ◽  
Alternatively Activated

Abstract Background In rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarisation into “classically” (M1) or “alternatively activated” (M2) phenotypes. In RA, CTLA4-Ig (abatacept) reduces the inflammatory activity of macrophages by interacting with the costimulatory molecule CD86. The study aimed to investigate the efficacy of CTLA4-Ig treatment to induce an M2 phenotype both in M1-polarised monocyte-derived macrophages (MDMs) obtained from healthy subjects (HS) and in cultured MDMs obtained from active RA patients. Methods Cultured MDMs were obtained from peripheral blood mononuclear cells of 7 active RA patients and from 10 HS after stimulation with phorbol myristate acetate (5 ng/mL) for 24 h. HS-MDMs were then stimulated with lipopolysaccharide (LPS, 1 mg/mL) for 4 h to induce M1-MDMs. M1-MDMs and RA-MDMs were treated with CTLA4-Ig (100 μM and 500 μM) for 3, 12, 24, and 48 h. The gene expression of CD80, CD86, and TLR4 (M1 markers); CD163, CD204, and CD206 (surface M2 markers); and MerTK (functional M2 marker) was evaluated by qRT-PCR. The protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by the Wilcoxon t-test. Results In LPS-induced HS-M1-MDMs, CTLA4-Ig 100 μM and 500 μM significantly downregulated the gene expression of M1 markers (3 h p<0.01 for all molecules; 12 h p<0.05 for TLR4 and CD86) and significantly upregulated that of M2 markers, primarily after 12 h of treatment (CD163: p < 0.01 and p < 0.05; CD206: p < 0.05 and p < 0.01; CD204: p < 0.05 by 100 mg/mL). Moreover, in these cells, CTLA4-Ig 500 μM increased the protein synthesis of surface M2 markers (p < 0.05). Similarly, in RA-MDMs, the CTLA4-Ig treatment significantly downregulated the gene expression of M1 markers at both concentrations primarily after 12 h (p < 0.05). Furthermore, both concentrations of CTLA4-Ig significantly upregulated the gene expression of CD206 (after 3 h of treatment; p < 0.05), CD163, and MerTK (after 12 h of treatment, p < 0.05), whereas CD204 gene expression was significantly upregulated by the high concentration of CTLA4-Ig (p < 0.05). The protein synthesis of all surface markers was increased primarily by CTLA4-Ig 500 μM, significantly for CD204 and CD206 after 24 h of treatment (p < 0.05). Conclusions CTLA4-Ig treatment seems to induce the in vitro shift from M1 to M2 macrophages, of both HS-M1-MDMs and RA-MDMs, as observed by the significant downregulation exerted on selected M1 markers and the upregulation of selected M2 markers suggesting an additional mechanism for its modulation of the RA inflammatory process.

Synergistic antitumor efficacy and altered gene expression signature in breast cancer cells treated with immunotoxins and cyclosporin A

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 13505-13505
Author(s):  
O. Fodstad ◽  
Y. Xi ◽  
K. Risberg ◽  
J. Ju ◽  
Y. G. Anderson
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Protein Synthesis ◽  
Breast Cancer Cells ◽  
Gene Expression Analysis ◽  
Synergistic Effects ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Gene Expression Signature

13505 Background: Immunotoxins (ITs) has shown limited clinical success related to liver toxicity and development of anti-IT antibodies. To delay the immune response we tested combinations of ITs and the Cyclosporin A (CsA). we have shown that one IT, currently in a phase I/II clinical trial, acts by inducing apoptosis and protein synthesis inhibition, but gene expression analysis of IT treated cells has never been reported. Hence, we also studied changes in gene expression induced by ITs alone and the effects of adding (CsA) on both treatment efficacy and gene expression signature. Methods: Human MA-11 breast cancer cells were treated in vitro with antiEGFR- and antiEPCAM-based ITs alone and in combinations with CsA. Therapeutic efficay was assessed by MTS cell viability assay. Total RNA from untreated and treated cells was isolated and CodeLink Uniset Human 20 k Oligo Bioarray (GE Healthcare, Amersham Biosciences, NJ), containing approximately 20,289 gene probes, was used to generate gene expression profiles. Gene expression analysis was carried out using GeneSpring software version 7.2 using One-way ANOVA with p<0.05. Comparisons of gene list across different groups were performed using Venn Diagrams. Results: Combination therapy produced remarkable synergistic effects in MA-11 cells in vitro and in metastasis models in vivo. Moreover, in conventional rats receiving repeated injections of ITs and CsA the formation of anti-IT antibodies was virtually abrogated. Changes in gene expression profiles induced by the ITs alone and in combination with CsA were evaluated to elucidate the underlying molecular mechanisms for the synergistic effects. The ITs each induced specific changes in expression of some apoptosis-related genes but also fogenes in pathways unrelated to apoptosis and protein synthesis. The addition of CsA induced up- or down-regulation of a number of interesting non-immune-associated genes Conclusions: Important shortcomings for successful clinical use of ITs may be overcome by combination therapy with CsA. The possibility for further improvement is provided by results of gene profiling studies identifying therapy-induced genes belonging to different cell signaling pathways. No significant financial relationships to disclose.

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