Green Fluorescent Protein Synthesis Research Articles

Cell‐Free Protein Synthesis in Bifunctional Hyaluronan Microgels: A Strategy for In Situ Immobilization and Purification of His‐Tagged Proteins

AbstractThe front cover artwork is provided by the Thiele group at Leibniz IPF Dresden (Germany). The image shows a microgel hosting cell‐free synthesis of green fluorescent protein. Despite its open, membrane‐free nature, the multifunctional microgel is also able to trap proteins inside its polymer matrix. Read the full text of the Article at 10.1002/syst.201900058.

Cell‐Free Protein Synthesis: A Platform Technology for Education

Hands‐on learning of transcription and translation remains limited due to a lack of classroom modules for early‐career students. Currently, classrooms implement animations, simulations, games, and bacterial transformation/protein expression to teach the genetic code. The cell wall poses the primary barrier for students to directly observe and manipulate the intracellular environment, limiting inquiry‐based learning of the genetic code. We have overcome this limit by levering transcription and translation in vitro. The open nature of cell‐free protein synthesis (CFPS) is achieved by removing the cell wall and genomic DNA, while isolating the cellular machinery involved in transcription and translation. This allows experimenter to harness the genetic code in a test tube and directly manipulate the reaction conditions for hands‐on learning. While reconstitution of cellular machinery in vitro for CFPS has been transformative for biotechnology, CFPS has not been accessible to early‐career students due to the complexity in reagent preparation and reaction set‐up. We have successfully adapted the CFPS biotechnology to develop a learning module accessible to early‐career STEM students focusing on learning objectives involving the genetic code. Here we report the reformulation of the CFPS reaction setup for simplicity and improved reagent shelf life in order to adapt CFPS for the classroom. First, our simplified platform requires the pipetting of only 3 pre‐mixed reagents, an 80% reduction in setup steps, making it more accessible to an early‐career student with little‐to‐no laboratory experience. Second, our simplified platform is shelf‐stable at −20°C rather than −80°C, making it accessible for storage and implementation in undergraduate and high school classrooms. Longer‐term experiments are in progress to validate shelf‐life at 4°C and room temperature. Our data show that these improvements are achieved without sacrificing CFPS performance in reaction kinetics or total protein yield. Third, our analysis of green fluorescent protein synthesis kinetics shows that 90% of maximum protein yields can be achieved within 3 hours at 37°C, making this platform compatible with most college laboratory courses. We believe that this educational technology will be transformative to the way the genetic code is taught in classrooms today. Toward this end, we have developed a CFPS classroom ‘kit’ for broad dissemination.Support or Funding InformationOur research is funded by the Center for Applications in Biotechnology @ Cal Poly SLO, Bill and Linda Frost Fund, and NSF‐1708919.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Stimulation of the Translation of Reporter mRNA in the Presence of Another mRNA in a Cell-Free System Based on Wheat Germ Extract

The translation of uncapped mRNAs encoding luciferase and green fluorescent protein in a cell-free translation system based on wheat germ extract has been studied. It turned out that two simultaneously translated (in one tube) different templates in a certain range of concentrations not only do not compete, but mutually enhance each other's translation. It has been shown that the synthesis of luciferase in the presence of mRNA that encodes green fluorescent protein is much more effective than in the translation of only luciferase mRNA at the same concentration. Similarly, the efficiency of the synthesis of green fluorescent protein increases in the presence of the template that encodes luciferase. It follows that the total effect of the concurrent translation of two different mRNAs exceeds the sum of the effects of the translation of each mRNA separately.

Exploiting the Protein Corona from Cell Lysate on DNA Functionalized Gold Nanoparticles for Enhanced mRNA Translation.

This study describes the use of DNA functionalized gold nanoparticles (AuNPs) to enhance the synthesis of proteins in cell lysate and examines the mechanisms behind the enhanced mRNA translation. With an appropriate DNA oligomer sequence that hybridizes to the 3'-untranslated region of two mRNA of interest, insulin and green fluorescent protein (GFP), we found that these DNA conjugated AuNPs (AuNP-DNA) introduced into HeLa cell lysate enhanced the synthesis of insulin and GFP by up to 2.18 and 1.80-fold, respectively, over baseline production with just the mRNA present. The insulin synthesis was markedly reduced with non-DNA citrate-capped AuNP (1.25-fold) and AuNP-DNA with a nonspecific poly(T) sequence (1.25-fold). We showed that both nonspecific adsorption of ribosomes and translation factors to form a lysate protein corona on AuNP-DNA and weak hybridization between DNA oligomers and mRNA of interest were important factors that brought translation factors, ribosomes, and mRNA into close proximity of each other. This could reduce the recycling time of ribosomes during mRNA translation, thereby increasing the efficiency of protein synthesis. The outcome of this work shows that with rational DNA design, it could be possible to modulate intracellular biological processes with AuNP-DNA and increase their production of proteins for various biomedical applications.

Dual effect of chloramphenicol peptides on ribosome inhibition.

Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.

Impact of pulsed light on cellular activity of Salmonella enterica.

The objective of this study was a comprehensive characterization of physiological changes of Salmonella enterica induced by intense broad spectrum pulsed light (PL). After exposing the bacteria to this nonthermal decontamination technology on a gel surface, multiple viability parameters beyond culturability were assessed. By applying flow cytometry, a luciferin-luciferase bioluminescence assay and a microplate assay to measure the current redox activity, the impact of pulsed light on the membrane potential, membrane integrity, esterase activity, efflux pump activity, expression of the green fluorescent protein (GFP), respiration activity and ATP-content of Salm.enterica ATCC BAA-1045 was determined. These culture-independent methods for assessing the bacterial activity were compared to the ability to grow on tryptic soy agar. It is shown that this strain is rather sensitive to PL considering colony count reductions, while on the other hand unculturable bacteria still exhibit significant cellular energetic functions. However, this residual activity after PL exposure significantly decreases during sample storage in buffer for 24h. This study also shows that the GFP expression of PL-treated cells which have rendered unculturable is severely reduced. This study reveals that although not all cellular functions of Salm.enterica are immediately shut down after PL exposure, the synthesis of new GFP is strongly reduced and affected to a similar extent as the culturability. It is shown for the first time, that even there is significant bacterial activity measurable after PL exposure, it is likely that nongrowing pathogenic bacteria like Salm.enterica are unable to express proteins, which is of great importance regarding their pathogenicity.

ChemInform Abstract: PCl3‐Mediated Synthesis of Green/cyan Fluorescent Protein Chromophores Using Amino Acids.

AbstractA convenient and rapid synthesis of green fluorescent protein (FP) chromophores (IV) and cyan FP chromophores (VI) starting from commercially available aromatic amino acids, acetyl chlorides, and anilines is described.

AmyI-1–18, a cationic α-helical antimicrobial octadecapeptide derived from α-amylase in rice, inhibits the translation and folding processes in a protein synthesis system

In our previous study, we used a cell-free rapid translation system (RTS), which is an invitro protein synthesis system based on Escherichia coli lysate, for evaluating the inhibition of green fluorescent protein (GFP) synthesis by pyrrhocoricin. In this study, using an RTS, we evaluated the inhibition of GFP synthesis by AmyI-1-18, an antimicrobial octadecapeptide. We found that, similarly to pyrrhocoricin, AmyI-1-18 inhibited GFP synthesis in the RTS in a concentration-dependent manner. In addition, the blockage of transcription and translation steps in the RTS was individually estimated using RT-PCR after gene expression to determine the mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that the inhibition of GFP synthesis by AmyI-1-18 did not occur at the transcription step but rather at the translation step. Furthermore, we assessed the inhibition of DnaK-mediated refolding of chemically denatured luciferase by AmyI-1-18; AmyI-1-18 inhibited the protein folding activity of the ATP-dependent DnaK/DnaJ molecular chaperone system in a concentration-dependent manner. Surface plasmon resonance (SPR) analysis showed that AmyI-1-18 strongly bound to RNA with a KD value of 1.4×10(-8)M but not to DNA and that AmyI-1-18 specifically bound to DnaK with a KD value of 4.4×10(-6)M. These SPR analysis results supported the results obtained in both the RTS and the molecular chaperone system. These results demonstrated that both RNA and DnaK are most likely the target of AmyI-1-18 in the protein synthesis system.

Genotoxicity of chemical compounds identification and assessment by yeast cells transformed with GFP reporter constructs regulated by the PLM2 or DIN7 promoter.

Yeast cells transformed with high-copy number plasmids comprising a green fluorescent protein (GFP)-encoding gene optimized for yeast under the control of the new DIN7 or PLM2 and the established RNR2 and RAD54 promoters were used to assess the genotoxic potential of chemical compounds. The activity of potential DNA-damaging agents was investigated by genotoxicity assays and by OxoPlate assay in the presence of various test compounds. The fluorescence signal generated by GFP in response to DNA damage was related to the different concentrations of analytes and the analyte-dependent GFP synthesis. The use of distinct DNA damage-inducible promoters presents alternative genotoxicity testing strategies by selective induction of promoters in response to DNA damage. The new DIN7 and PLM2 systems show higher sensitivity than the RNR2 and RAD54 systems in detecting 4-nitroquinoline-N-oxide and actinomycin D. Both DIN7 and PLM2 systems are able to detect camptothecin while RNR2 and RAD54 systems are not. Automated laboratory systems with assay performance on 384-well microplates provide for cost-effective high-throughput screening of DNA-damaging agents, reducing compound consumption to about 53% as compared with existing eukaryotic genotoxicity bioassays.

Genomic analysis reveals distinct concentration-dependent evolutionary trajectories for antibiotic resistance in Escherichia coli.

Evolution of bacteria under sublethal concentrations of antibiotics represents a trade-off between growth and resistance to the antibiotic. To understand this trade-off, we performed in vitro evolution of laboratory Escherichia coli under sublethal concentrations of the aminoglycoside kanamycin over short time durations. We report that fixation of less costly kanamycin-resistant mutants occurred earlier in populations growing at lower sublethal concentration of the antibiotic, compared with those growing at higher sublethal concentrations; in the latter, resistant mutants with a significant growth defect persisted longer. Using deep sequencing, we identified kanamycin resistance-conferring mutations, which were costly or not in terms of growth in the absence of the antibiotic. Multiple mutations in the C-terminal end of domain IV of the translation elongation factor EF-G provided low-cost resistance to kanamycin. Despite targeting the same or adjacent residues of the protein, these mutants differed from each other in the levels of resistance they provided. Analysis of one of these mutations showed that it has little defect in growth or in synthesis of green fluorescent protein (GFP) from an inducible plasmid in the absence of the antibiotic. A second class of mutations, recovered only during evolution in higher sublethal concentrations of the antibiotic, deleted the C-terminal end of the ATP synthase shaft. This mutation confers basal-level resistance to kanamycin while showing a strong growth defect in the absence of the antibiotic. In conclusion, the early dynamics of the development of resistance to an aminoglycoside antibiotic is dependent on the levels of stress (concentration) imposed by the antibiotic, with the evolution of less costly variants only a matter of time.

Metabolic flux distribution and thermodynamic analysis of green fluorescent protein production in recombinant Escherichia coli: The effect of carbon source and CO 2 partial pressure

Increasing recombinant protein production yields from bacterial cultures remains an important challenge in biotechnology. Acetate accumulation due to high dissolved carbon dioxide (pCO2) concentrations in the medium has been identified as a factor that negatively affects such yields. Under appropriate culture conditions, acetate could be re-assimilated by bacterial cells to maintain heterologous proteins production. In this work, we developed a simplified metabolic network aiming to establish a reaction rate analysis for a recombinant Escherichia coli when producing green fluorescent protein (GFP) under controlled pCO2 concentrations. Because E. coli is able to consume both glucose and acetate, the analysis was performed in two stages. Our results indicated that GFP synthesis is an independent process of cellular growth in some culture phases. Additionally, recombinant protein production is influenced by the available carbon source and the amount of pCO2 in the culture medium. When growing on glucose, the increase in the pCO2 concentration produced a down-regulation of central carbon metabolism by directing the carbon flux toward acetate accumulation; as a result, cellular growth and the overall GFP yield decreased. However, the maximum specific rate of GFP synthesis occurred with acetate as the main available carbon source, despite the low activity in the other metabolic pathways. To maintain cellular functions, including GFP synthesis, carbon flux was re-distributed toward the tricarboxylic acid cycle and the pentose phosphate pathway to produce ATP and NADH. The thermodynamic analysis allowed demonstrating the feasibility of the simplified network for describing the metabolic state of a recombinant system.

A low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors

BackgroundMicrobial cell population heterogeneity is now recognized as a major source of issues in the development and optimization of bioprocesses. Even if single cell technologies are available for the study of microbial population heterogeneity, only a few of these methods are available in order to study the dynamics of segregation directly in bioreactors. In this context, specific interfaces have been developed in order to connect a flow cytometer directly to a bioreactor for automated analyses. In this work, we propose a simplified version of such an interface and demonstrate its usefulness for multiplexed experiments.ResultsA low-cost automated flow cytometer has been used in order to monitor the synthesis of a destabilized Green Fluorescent Protein (GFP) under the regulation of the fis promoter and propidium iodide (PI) uptake. The results obtained showed that the dynamics of GFP synthesis are complex and can be attributed to a complex set of biological parameters, i.e. on the one hand the release of protein into the extracellular medium and its uptake modifying the activity of the fis promoter, and on the other hand the stability of the GFP molecule itself, which can be attributed to the protease content and energy status of the cells. In this respect, multiplexed experiments have shown a correlation between heat shock and ATP content and the stability of the reporter molecule.ConclusionThis work demonstrates that a simplified version of on-line FC can be used at the process level or in a multiplexed version to investigate the dynamics of complex physiological mechanisms. In this respect, the determination of new on-line parameters derived from automated FC is of primary importance in order to fully integrate the power of FC in dedicated feedback control loops.

Broadening Horizons and Teaching Basic Biology Through Cell-Free Synthesis of Green Fluorescent Protein in a High School Laboratory Course

Cell-free protein synthesis (CFPS) has emerged as a practical method for producing a broad variety of proteins. In addition, the direct accessibility to the reaction environment makes CFPS particularly suitable as a learning vehicle for fundamental biological concepts. Here, we describe its implementation as a teaching tool for a high school laboratory course. Ninety students in a biotechnology class used CFPS to study the effects of the concentrations of amino acids, cell extract, DNA, and the energy source on accumulation of active super-folder green fluorescent protein. Students estimated product concentrations simply by comparing solution colors to a printed green color gradient. This simple and inexpensive method allows for immediate measurements, and 26 of the 30 groups observed measurable product concentrations within 60 min. These student-generated data were then discussed to illustrate concepts of data analysis such as outliers and standard deviation. We also combined the laboratory experience with a visit to a university campus that included a laboratory tour and a college-style lecture. Our overall objective was to excite the students about the scientific enterprise and to instill a sense of personal relevance and attainability so that these students could realistically consider technical careers.

Hot-wiring cells

Hot-wiring cells

Light‐Addressable Capsules as Caged Compound Matrix for Controlled Triggering of Cytosolic Reactions

Release me: polyelectrolyte capsules with different cargo in their cavities and plasmonic and magnetic nanoparticles in their walls were synthesized. Enzymatic reactions were triggered inside cells by light-mediated opening of two individual capsules containing either an enzyme or its substrate, by using photothermal heating. Furthermore, this technique allows controlled release of mRNA from capsules, thereby resulting in synthesis of green fluorescent protein (GFP).

Translocator protein (Tspo) gene promoter-driven green fluorescent protein synthesis in transgenic mice: an in vivo model to study Tspo transcription

Translocator protein (TSPO), previously known as the peripheral-type benzodiazepine receptor, is a ubiquitous drug- and cholesterol-binding protein primarily found in the outer mitochondrial membrane as part of a mitochondrial cholesterol transport complex. TSPO is present at higher levels in steroid-synthesizing and rapidly proliferating tissues and its biological role has been mainly linked to mitochondrial function, steroidogenesis and cell proliferation/apoptosis. Aberrant TSPO levels have been linked to multiple diseases, including cancer, endocrine disorders, brain injury, neurodegeneration, ischemia-reperfusion injury and inflammatory diseases. Investigation of the functions of this protein in vitro and in vivo have been mainly carried out using high-affinity drug ligands, such as isoquinoline carboxamides and benzodiazepines and more recently, gene silencing methods. To establish a model to study the regulation of Tspo transcription in vivo, we generated a transgenic mouse model expressing green fluorescent protein (GFP) from Aequorea coerulescens under control of the Tspo promoter region (Tspo-AcGFP). The expression profiles of Tspo-AcGFP, endogenous TSPO and Tspo mRNA were found to be well-correlated. Tspo-AcGFP synthesis in the transgenic mice was seen in almost every tissue examined and as with TSPO in wild-type mice, Tspo-AcGFP was highly expressed in steroidogenic cells of the endocrine and reproductive systems, epithelial cells of the digestive system, skeletal muscle and other organs. In summary, this transgenic Tspo-AcGFP mouse model recapitulates endogenous Tspo expression patterns and could be a useful, tractable tool for monitoring the transcriptional regulation and function of Tspo in live animal experiments.

Cell-free protein synthesis and assembly on a biochip

Biologically active complexes such as ribosomes and bacteriophages are formed through the self-assembly of proteins and nucleic acids. Recapitulating these biological self-assembly processes in a cell-free environment offers a way to develop synthetic biodevices. To visualize and understand the assembly process, a platform is required that enables simultaneous synthesis, assembly and imaging at the nanoscale. Here, we show that a silicon dioxide grid, used to support samples in transmission electron microscopy, can be modified into a biochip to combine in situ protein synthesis, assembly and imaging. Light is used to pattern the biochip surface with genes that encode specific proteins, and antibody traps that bind and assemble the nascent proteins. Using transmission electron microscopy imaging we show that protein nanotubes synthesized on the biochip surface in the presence of antibody traps efficiently assembled on these traps, but pre-assembled nanotubes were not effectively captured. Moreover, synthesis of green fluorescent protein from its immobilized gene generated a gradient of captured proteins decreasing in concentration away from the gene source. This biochip could be used to create spatial patterns of proteins assembled on surfaces.

Cell-Free Protein Synthesis inside Giant Unilamellar Vesicles Analyzed by Flow Cytometry

Lipid vesicles have been used as model cell systems, in which an in-vitro transcription-translation system (IVTT) is encapsulated to carry out intravesicular protein synthesis. Despite a large number of previous studies, a quantitative understanding of how protein synthesis inside the vesicles is affected by the lipid membrane remains elusive. This is mainly because of the heterogeneity in structural properties of the lipid vesicles used in the experiments. We investigated the effects of the phospholipid membrane on green fluorescent protein (GFP) synthesis occurring inside cell-sized giant unilamellar vesicles (GUV), which have a defined quantity of lipids relative to the reaction volume. We first developed a method to distinguish GUV from multilamellar vesicles using flow cytometry (FCM). Using this method, we investigated the time course of GFP synthesis using one of the IVTT, the PURE system, and found that phospholipid in the form of GUV has little effect on GFP synthesis based on three lines of investigation. (1) GFP synthesis inside the GUV was not dependent on the size of GUV (2) or on the fraction of cholesterol or anionic phospholipid constituting the GUV, and (3) GFP synthesis proceeded similarly in GUV and in the test tube. The present results suggest that GUV provides an ideal reaction environment that does not affect the internal biochemical reaction. On the other hand, we also found that internal GFP synthesis is strongly dependent on the chemical composition of the outer solution.

A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression

Here we report on the construction of a tetracycline inducible expression vector that allows a tightly regulable gene expression in Corynebacterium glutamicum which is used in industry for production of small molecules such as amino acids. Using the green fluorescent protein (GFP) as a reporter protein we show that this vector, named pCLTON1, is characterized by tight repression under non-induced conditions as compared to a conventional IPTG inducible expression vector, and that it allows gradual GFP synthesis upon gradual increase of anhydrotetracycline addition.

Polysaccharide nanogel–cyclodextrin system as an artificial chaperone for in vitro protein synthesis of green fluorescent protein

Polysaccharide nanogels have been demonstrated to aid the refolding processes of chemically or thermally denatured proteins, a function that is similar to that of natural molecular chaperones. In this study, we examined the possibilities of using the nanogel chaperone system to mediate protein folding in a cell-free (in vitro) protein synthesis system containing transcription/translation factors. High-performance liquid chromatography showed that a polysaccharide nanogel comprising cholesteryl group-bearing pullulan (CHP) trapped unfolded or partially folded green fluorescent protein (GFP) expressed in the cell-free system. The protein release and refolding processes, which are induced by ATP in natural molecular chaperone systems, were also simulated by methyl-β-cyclodextrin (M-β-CD). The CHP nanogels dissociate on complexation with M-β-CD to yield dissociated CHP. Thus, the dissociation of the CHP nanogel–protein complex subsequently allows for the release and folding of GFP. The folding kinetics in the presence of the CHP nanogel and M-β-CD was comparable to that of spontaneous folding in the absence of CHP/M-β-CD, indicating that the CHP nanogels did not affect protein synthesis in the cell-free system, providing correctly folded active proteins. The molecular chaperone function of polysaccharide nanogel was demonstrated for the folding of newly synthesized green fluorescent protein (GFP) in a cell-free protein synthesis system. Nanogel comprising cholesteryl group-bearing pullulan (CHP) trapped unfolded or partially folded GFP expressed in the cell-free system. The structure of the CHP nanogel was disrupted by the addition of cyclodextrins, and the protein complexed with the nanogel was released and folded into the mature form.