The authors would like to acknowledge the following research support: EPSRC [EP/K038648/1] for SJM as a PDRA with PSF; Wellcome Trust sponsored ISSF fellowship for SJM with PSF at Imperial College London; Royal Society research grant [RGS\R1\191186]; Wellcome Trust SEED award [217528/Z/19/Z] for SJM at the University of Kent; and Global Challenges Research Fund (GCRF) Ph.D. scholarship for KC at the University of Kent.

NOTE: See Table 1 and Table 2 for recipes for GYM medium and agar plate and S30A and S30B wash buffers.
1. Preparation of solutions and general guidance
<ol>
	<li>Keep all solutions, cells (post-growth), and cell extracts on ice after preparation, unless an exception is stated.</li>
	<li>Store stocks for 1 M Mg-glutamate, 4 M K-glutamate, 40% (w/v) PEG 6000, 1&#160;g/mL polyvinylsulfonic acid at room temperature, and all other stocks at -80 &#176;C. Minimize...

In this manuscript, a high-yield S. venezuelae TX-TL protocol has been described with detailed steps that are straightforward to conduct for both experienced and new users of TX-TL systems. Several features from existing Streptomyces45 and E. coli TX-TL41 protocols have been removed to establish a minimal, yet high-yield protocol for S. venezuelae TX-TL5,26. The workflow recommend...

Cell-free transcription-translation (TX-TL) systems provide an ideal prototyping platform for synthetic biology to implement rapid design-build-test-learn cycles, the conceptual engineering framework for synthetic biology1. In addition, there is growing interest in TX-TL systems for high-value recombinant protein production in an open-reaction environment2, for example, to incorporate non-standard amino acids in antibody-drug conjugates3. Specifically, TX-TL requires a cell extract, plasmid or linear DNA, and an energy solution to catalyze protein synthesis in batch or semicontinuous reactions. While Escherichia coli TX-TL is the dominant cell-free system, a number of emerging non-model TX-TL systems have attracted attention for different applications4,5,6,7,8. Key advantages of TX-TL include flexible scalability (nanoliter to liter scale)9,10, strong reproducibility, and automated workflows8,11,12. In particular, automation of TX-TL permits the accelerated characterization of genetic parts and regulatory elements8,12,13.
In terms of reaction setup, TX-TL requires both primary and secondary energy sources, as well as amino acids, cofactors, additives, and a template DNA sequence. Nucleotide triphosphates (NTPs) provide the primary energy source to drive initial mRNA (ATP, GTP, CTP, and UTP) and protein synthesis (only ATP and GTP). To increase TX-TL yields, NTPs are regenerated through the catabolism of a secondary energy source, such as maltose14, maltodextrin15, glucose14, 3-phosphoglycerate (3-PGA)16, phosphoenolpyruvate17, and L-glutamate18. This inherent metabolic activity is surprisingly versatile, yet poorly studied, especially in emerging TX-TL systems. Each energy source has distinct properties and advantages in terms of ATP yield, chemical stability, and cost, which is an important consideration for scaled-up TX-TL reactions. So far, current protocols for E. coli TX-TL have reached up to 4.0 mg/mL (~157 &#181;M) for the model green fluorescent protein (GFP), using a blend of 3-PGA (30 mM), maltodextrin (60 mM), and D-ribose (30 mM) as the secondary energy source19.
Recently, there has been a rising interest in studying secondary metabolite biosynthetic pathways in TX-TL systems20,21,22. Specifically, Actinobacteria are a major source of secondary metabolites, including antibiotics and agricultural chemicals23,24. Their genomes are enriched with so-called biosynthetic gene clusters (BGCs), which encode enzymatic pathways for secondary metabolite biosynthesis. For the study of Actinobacteria genetic parts and biosynthetic pathways, a range of Streptomyces-based TX-TL systems have recently been developed5,6,25,26. These specialized Streptomyces TX-TL systems are potentially beneficial for the following reasons: [1] provision of a native protein folding environment for enzymes from Streptomyces spp.26; [2] access to an optimal tRNA pool for high G+C (%) gene expression; [3] active primary metabolism, which potentially can be hijacked for the supply of biosynthetic precursors; and [4] provision of enzymes, precursors, or cofactors from secondary metabolism present in the native cell extract.&#160;Hence, a high-yield S.venezuelae TX-TL toolkit has recently been established to harness these unique capabilities5.
Streptomyces venezuelae is an emerging host for synthetic biology with a rich history in industrial biotechnology5,27,28,29 and as a model system for studying cell division and genetic regulation in Actinobacteria30,31,32.&#160;The main type strain, S. venezuelae ATCC 10712, has a relatively large genome of 8.22 Mb with 72.5% G+C content (%) (Accession number: CP029197), which encodes 7377 coding sequences, 21 rRNAs, 67 tRNAs, and 30 biosynthetic gene clusters27. In synthetic biology, S. venezuelae ATCC 10712 is an attractive chassis for the heterologous expression of biosynthetic pathways. Unlike most other Streptomyces stains, it provides several key advantages, including a rapid doubling time (~40 min), an extensive range of genetic and experimental tools5,28, lack of mycelial clumping, and sporulation in liquid media28,33. Several studies have also demonstrated the use of S. venezuelae for heterologous production of a diverse array of secondary metabolites, including polyketides, ribosomal and nonribosomal peptides34,35,36,37,38. These combined features make this strain an attractive microbial host for synthetic biology and metabolic engineering applications. While S. venezuelae is not the dominant Streptomyces model for heterologous gene expression, with further developments, it is primed for broader use within natural product&#160;discovery.
This manuscript presents a detailed protocol (Figure 1) for a high-yield S. venezuelae TX-TL system, which has been updated from the original previously-published protocol26. In this work, the energy solution and reaction conditions have been optimized to increase protein yield up to 260 &#956;g/mL for the mScarlet-I reporter protein in a 4 h, 10 &#956;L batch reaction, using a standard plasmid, pTU1-A-SP44-mScarlet-I. This plasmid has been specifically designed to enable various methods of detecting protein expression. The protocol is also streamlined, while the energy system has been optimized to reduce the complexity and cost of setting up cell-free reactions without compromising the yield. Along with the optimized TX-TL system, a library of genetic parts has been developed for fine-tuning gene expression and as fluorescent tools for monitoring TX-TL in real time, thereby creating a versatile platform for prototyping gene expression and natural product biosynthetic pathways from Streptomyces spp. and related Actinobacteria.
In this work, the recommended standard plasmid (pTU1-A-SP44-mScarlet-I) can be used to establish the S. venezuelae TX-TL workflow in a new laboratory and is available on AddGene (see Supplemental Table S1). pTU1-A-SP44-mScarlet-I provides the user with the flexibility to study other open-reading frames (ORFs). The mScarlet-I ORF is codon-optimized for S. venezuelae gene expression. The SP44 promoter is a strong constitutive promoter that is highly active in both E. coli and Streptomyces spp.39. The plasmid has two unique restriction enzyme sites (NdeI, BamHI) to allow the sub-cloning of new ORFs in-frame with a joint C-terminal FLAG-tag and fluorescein arsenical hairpin (FlAsH) binder tag system. Alternatively, both tags can be removed with the inclusion of a stop codon after sub-cloning a new gene. With this base vector, the high-yield expression of a range of proteins has been demonstrated, namely proteins from the oxytetracycline biosynthesis pathway and an uncharacterized nonribosomal peptide synthetase (NRPS) from Streptomyces rimosus (Figure 2). In terms of mRNA detection, the pTU1-A-SP44-mScarlet-I standard plasmid contains a dBroccoli aptamer (in the 3&#39;-untranslated region) for detection with the 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) probe. For increased flexibility, a toolset of EcoFlex40-compatible MoClo parts has also been made available on AddGene, including an EcoFlex-compatible Streptomyces shuttle vector (pSF1C-A-RFP/pSF2C-A-RFP) and a range of pTU1-A-SP44 variant plasmids expressing superfolder green fluorescence protein (sfGFP), mScarlet-I, mVenus-I, and &#946;-glucuronidase (GUS). In particular, the pSF1C-A plasmid is derived from pAV-gapdh28 and is cured of BsaI/BsmBI sites for MoClo assembly. pSF1C-A-RFP/pSF2C-A-RFP is equivalent to pTU1-A-RFP/pTU2-A-RFP from EcoFlex40 but contains additional functionality for conjugation and chromosomal integration in Streptomyces spp. using the phiC31 integrase system28.
The first stage of the protocol involves the growth of the S. venezuelae ATCC 10712 or a closely related strain, cell harvest at mid-exponential phase, cell wash steps, and equilibration in S30A and S30B buffers. This stage requires three days, and the time for cell growth can be used to prepare the remaining components as described below. The harvested cells are then lysed by sonication, clarified, and undergo a run-off reaction. At this final stage of preparation, the cell extracts can be prepared for long-term storage at -80 &#176;C to minimize loss of activity. For the assembly of TX-TL reactions using this protocol, a Streptomyces Master Mix (SMM) is presented, with the option of a Minimal Energy Solution format (MES) that gives comparable yields. Further, it is recommended to streak a fresh culture of S. venezuelae ATCC 10712 from a -80 &#176;C glycerol stock onto a GYM agar plate and incubate at 28 &#176;C for at least 48-72 h until single colonies are visible. Only fresh cultures should be used for the following steps.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.5 L UltraYield Flask</td>
			<td>Thomson</td>
			<td>931136-B</td>
			<td></td>
		</tr>
		<tr>
			<td>3-PGA (&#62;93%)</td>
			<td>Sigma</td>
			<td>P8877</td>
			<td></td>
		</tr>
		<tr>
			<td>384 Well Black/Clear Bottom Plate</td>
			<td>ThermoFisher</td>
			<td>10692202</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride (98%)</td>
			<td>Fluorochem</td>
			<td>44722</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP, CTP, UTP, GTP (100 mM solution, &#62;99%)</td>
			<td>ThermoFisher</td>
			<td>R0481</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin (contact supplier for purity)</td>
			<td>Melford</td>
			<td>C46000-25.0</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-glucose (contact supplier for purity)</td>
			<td>Melford</td>
			<td>G32040</td>
			<td></td>
		</tr>
		<tr>
			<td>DFHBI (&#8805;98% - HPLC)</td>
			<td>Sigma</td>
			<td>SML1627</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (contact supplier for purity)</td>
			<td>Melford</td>
			<td>MB1015</td>
			<td></td>
		</tr>
		<tr>
			<td>FlAsH-EDT2 (contact supplier for purity)</td>
			<td>Santa Cruz Biotech</td>
			<td>sc-363644</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose-6-phosphate (&#62;98%)</td>
			<td>Sigma</td>
			<td>G7879</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Free Acid (contact supplier for purity)</td>
			<td>Melford</td>
			<td>B2001</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamic acid hemimagnesium salt tetrahydrate (&#62;98%)</td>
			<td>Sigma</td>
			<td>49605</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride (98%)</td>
			<td>Fluorochem</td>
			<td>494356</td>
			<td></td>
		</tr>
		<tr>
			<td>Malt extract</td>
			<td>Sigma</td>
			<td>70167-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-6000</td>
			<td>Sigma</td>
			<td>807491</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 96-well Microdialysis Plate, 10K MWCO</td>
			<td>ThermoFisher</td>
			<td>88260</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(vinyl sulfate) potassium salt</td>
			<td>Sigma</td>
			<td>271969</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium glutamate (&#62;99%)</td>
			<td>Sigma</td>
			<td>G1149</td>
			<td></td>
		</tr>
		<tr>
			<td>RTS amino acid sampler</td>
			<td>5 Prime</td>
			<td>2401530</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (99%)</td>
			<td>Fluorochem</td>
			<td>94554</td>
			<td></td>
		</tr>
		<tr>
			<td>Supelclean LC-18 SPE C-18 SPE column (1 g)</td>
			<td>Sigma</td>
			<td>505471</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Melford</td>
			<td>Y1333</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platereader</td>
			<td>BMG</td>
			<td>Omega</td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-yield streptomyces transcription-translation toolkit for synthetic biology and natural product applications

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate&#160;as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 &#956;L) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 &#956;M) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput&#160;gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.

This detailed protocol is provided as an example to help the user establish a Streptomyces TX-TL system based on the S. venezuelae ATCC 10712 model strain (Figure 1). The user may seek to study other Streptomyces strains; however, the growth/harvesting stages of other strains with longer doubling times or distinct&#160;growth preferences will need to be custom-optimized to achieve peak results. For the representative result, the mScarlet-I fluorescent protein from ...

Watch this Scientific Journal Video about A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications at JoVE.com

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

This protocol details an enhanced method for synthesizing high yields of recombinant proteins from a Streptomyces venezuelae cell-free transcription-translation (TX-TL) system.

A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a ...

Our protocol provides a simplified workflow for Streptomyces venezuelae cell-free transcription-translation. Our system harnesses the metabolic enzymes and pathways from this non-model organism. The main advantages are the heterologous expression of genes with high GC content, the provision of a favorable protein forwarding environment, and an open system for fine-tuning of biosynthetic pathways.Our method will open new opportunities into studying Streptomyces and related high GC bacteria. It allows to study enzymes and gene expression in these bacteria. This technique has been developed for ease of implementation by new users.The only step that requires prior experience or familiarization is sonication to produce highly active crude extracts. To begin harvesting the pre-cultured cells on the third day, dilute the overnight culture at the ratio of one-to-10 with a fresh GYN medium for the optical density or OD measurement at 600 nanometers. If the OD of the culture at 600 nanometers is less than 0.3, increase the shaking speed to 250 to 300 rotations per minute and grow until an OD reaches 0.3.Grow for no longer than an additional two hours. If OD is greater than 0.3, transfer the cultures to centrifugation containers and rapidly cool on wet ice for 30 minutes. While waiting for the cell culture to cool, prepare four milliliters of fresh one molar dithiothreitol, S30A and S30B buffers and keep them on ice.After weighing an empty 50 milliliter centrifuge tube, pre-chill it at minus 20 degrees Celsius. Add two milliliters of one molar dithiothreitol to one liter of S30A buffer on ice and mix well. Centrifuge the cells at 6, 000 times G for 10 minutes at four degrees Celsius.After discarding the supernatant in a quick motion, add 250 milliliters of S30A buffer and resuspend the cells by shaking the centrifugation bottles vigorously until the cell clumps are homogeneously dispersed. Then centrifuge the cells again for six minutes. After centrifugation, discard the supernatant and repeat the addition of S30A buffer, followed by centrifugation as demonstrated.Next, add two milliliters of one molar DTT to one liter of S30B buffer on ice and mix well. Add 250 milliliters of S30B buffer to the cells and repeat centrifugation. Resuspend the cell pellet in 10 milliliters of S30B buffer and transfer the cell suspension to the pre-weighed pre-chilled 50 milliliter centrifuge tube.If required, transfer the residual cells with an additional five to 10 milliliters of S30B buffer and fill the tube to 50 milliliters volume with S30B. Centrifuge the cells for 10 minutes. Discard the supernatant and repeat the centrifugation at previous settings as demonstrated.After discarding the supernatant, aspirate the remaining S30B supernatant with a 100 to 200 microliter pipette and weigh the wet cell pellet. For every one gram of wet cells, add 0.9 milliliters of S30B buffer. Resuspend the cells using either a Pasteur pipette or vortex.Centrifuge briefly for about 10 seconds up to 500 G to sediment the cells. Lower the sonicator tip into the cell suspension until the tip is about one centimeter below the liquid surface. Input different parameters in the sonicator settings.Set frequency to 20 kilohertz, amplitude to 65%pulse on and off time to 10 seconds, and total sonication time to one minute. Begin sonication and move the tube up or down and sideways during first two resting cycles to ensure the cells are evenly sonicated. If the cells are not fully lysed, invert the tube two to three times and repeat the sonication for an additional one or two 10-second cycles with frequent mixing until the cells are fully disrupted.Centrifuge the lysed cells to remove the cell debris, then transfer the supernatant into a 1.5 milliliter microcentrifuge tube as one milliliter aliquot. For the runoff reaction of the cell extracts, incubate the cell containing the cell extract at 30 degrees Celsius for 60 minutes on a heat block or in the incubator without a shaker. For the transcription-translation reaction, thaw the cell extract SMM or MES solution and plasmid DNA on ice.Pre-chill a 384-well plate at minus 20 degrees Celsius. Set up the transcription-translation reaction on the ice where 25%of the volume is plasmid DNA, 33.33%is cell extract and 41.67%is SMM solution. Gently vortex the mixture for about five seconds at a low speed setting to ensure the solution is homogenous while avoiding foaming or bubble formation.Transfer three aliquots of 10 microliters into three wells of a 384-well plate as a technical triplicate without introducing air bubbles. After sealing the plate with a transparent cover, spin the plate at 400 times G for five seconds and incubate the reaction at 28 degrees Celsius either in an incubator or a plate reader without shaking. Streptomyces venezuelae cell-free transcription-translation was optimized for high yield expression of fluorescent proteins from the pTU1-A-SP44 standard plasmid, as well as biosynthetic enzymes detected by gel electrophoresis.The expression of the pTU1-A-SP44-mScarlet-I standard plasmid was measured using several methods. The real-time fluorescence measurements of mRNA using the dBroccoli RNA aptamer, immature mScarlet-I protein using the FlAsH tag system, and mature mScarlet-I protein were carried out. The in-gel fluorescence staining of mScarlet-I using FlAsH tag was conducted in addition to the Coomassie Blue staining of the total cell-free protein.Following this procedure, activity assays can be performed to study enzymes and pathways that are challenging using standard methods. It also extends towards scaled up biosynthesis and high throughput gene expression. We are developing this technique to study metabolic enzymes from high GC bacteria.We have shown the potential of our system to study scaled up biosynthesis in cell-free reactions.

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3.9K vistas.  South Kensington Campus. Nuestro protocolo proporciona un flujo de trabajo simplificado para Streptomyces venezuelae transcripción-traducción libre de células. Nuestro sistema aprovecha las enzimas metabólicas y las vías de este organismo no modelo. Las principales ventajas son la expresión heteróloga de genes con alto contenido de GC, la provisión de un entorno favorable de reenvío de proteínas y un sistema abierto para el ajuste fino de las vías biosintéticas.Nuestro método abrirá nuevas oportu...