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 the number of freeze-thaw cycles to avoid chemical degradation.</li>
	<li>For the preparation of energy solution stocks (see Table 3) such as 3-PGA (requires pH adjustment), follow the guidance provided in the E. coli TX-TL protocol41. 
	NOTE: All components are fully soluble in ddH2O and stored as aliquots in the -80 &#176;C freezer.</li>
	<li>Defrost individual stocks or energy solutions (described later) on ice. Heat the amino acids stock at 42 &#176;C with vortexing for ~15-30 min to solubilize all amino acids.</li>
	<li>As some amino acids (L-Cys, L-Tyr, L-Leu) precipitate on ice, while minimizing rest time, leave this solution at room temperature and use a vortex to dissolve.</li>
	<li>Add the calculated volumes (Table 3) of stock solutions and water and mix well using a vortex.</li>
	<li>Aliquot the energy solution as 20-100 &#181;L aliquots per tube, or as desired, on ice and store at -80 &#176;C until further use.</li>
</ol>
2. Preparation of S. venezuelae ATCC 10712 cells
<ol>
	<li>Day 1-Media/buffer preparation and overnight pre-culture
	<ol>
		<li>Prepare 1 L of sterile GYM liquid medium in a 2 L baffled flask, as described in Table 1. See the Table of Materials for equipment/chemical/reagent sources.</li>
		<li>Prepare 1 x 50 mL of sterile GYM liquid medium in a 250 mL Erlenmeyer flask, as described in Table 1.</li>
		<li>Prepare 100 mL of 1 M HEPES-KOH pH 7.5, 100 mL of 1 M MgCl2, and 500 mL of 4 M NH4Cl solutions to make 1 L of S30A and 1 L of S30B wash buffers. See Table 2 for the recipes.</li>
		<li>Prepare the overnight pre-culture. Pre-warm the sterile 50 mL of GYM liquid medium in a 250 mL Erlenmeyer flask to 28 &#176;C for 30 min.</li>
		<li>Inoculate a single colony of S. venezuelae ATCC 10712 (or related strain) from a GYM agar plate into prewarmed 50 mL of GYM liquid medium and incubate at 28 &#176;C, 200 rpm for 16 h (overnight pre-culture).</li>
	</ol>
	</li>
	<li>Day 2-Prepare daytime pre-culture and main growth culture.
	<ol>
		<li>Pre-warm 50 mL of sterile GYM liquid medium in a 250 mL Erlenmeyer flask at 28 &#176;C for 30 min.</li>
		<li>Transfer 1 mL of overnight pre-culture into pre-warmed 50 mL of GYM liquid medium and incubate at 28 &#176;C, 200 rpm for 8 h (daytime pre-culture).</li>
		<li>After this growth period, check the OD600 in a spectrophotometer using a 1:10 dilution with sterile GYM medium in a 1 mL (1 cm path length) plastic cuvette. 
		NOTE: The OD600 should have reached at least 3-4. If there is poor growth, it is advisable to repeat steps 2.2.1-2.2.2.</li>
		<li>Sub-culture 0.25 mL of daytime pre-culture into 1 L of liquid GYM medium in 2 L baffled flasks.</li>
		<li>Shake overnight at 28 &#176;C, 200 rpm for 14 h.</li>
	</ol>
	</li>
	<li>Day 3-Harvest cells
	<ol>
		<li>After the previous incubation period (14 h), record the OD600 of the main culture. Dilute the overnight culture 1:10 with fresh GYM medium for OD600 measurement. 
		NOTE: The OD600 should have reached 3.0-4.0 at this stage.</li>
		<li>If OD600&#60;3.0, increase the shaking speed to 250-300 rpm and grow until an OD600 of 3.0 is reached. Grow for no longer than an additional 2 h (16 h in total).</li>
		<li>If OD600&#62;3.0, transfer the cultures to centrifugation containers and rapidly cool on wet ice for 30 min.</li>
		<li>While waiting for the cell culture to cool on ice, prepare 4 mL of fresh 1 M dithiothreitol (DTT), S30A and S30B buffers, as described in Table 1, and keep them on ice. See the Table of Materials for chemical/reagent source.</li>
		<li>Pre-weigh an empty 50 mL centrifuge tube and pre-chill at -20 &#176;C.</li>
		<li>Add 2 mL of 1 M DTT to 1 L of S30A buffer on ice and mix well. 
		NOTE: Add DTT to the S30A and S30B wash buffers only before using them.</li>
		<li>Centrifuge cells at 6,000 &#215; g, 4 &#176;C, 10 min, and carefully discard the supernatant in a quick and single motion. 
		NOTE: If the pellet is disturbed, maximize cell retention with residual GYM medium and continue the protocol.</li>
		<li>Add 500 mL of S30A buffer and resuspend the cells by shaking the centrifugation bottles vigorously until the cell clumps are homogeneously dispersed.</li>
		<li>Centrifuge the cells at 6,000 &#215; g, 4 &#176;C, 6 min, and carefully discard the supernatant. 
		NOTE: Although the cell pellet will be firmer at this point, some cells will remain in suspension (see Figure 1). Treat as described in 2.3.7 and retain as many cells as possible.</li>
		<li>Repeat steps 2.3.8-2.3.9.</li>
		<li>Add 2 mL of 1 M DTT to 1 L of S30B buffer on ice and mix well. Add 500 mL of S30B buffer to the cells. Repeat step 2.3.9.</li>
		<li>Resuspend the cell pellet in 10 mL of S30B buffer and transfer to the pre-weighed, pre-chilled 50 mL centrifuge tube. If required, transfer the residual cells with an additional 5-10 mL of S30B buffer. Fill to 50 mL with S30B.</li>
		<li>Centrifuge cells at 6,000 &#215; g, 4 &#176;C, 10 min, and carefully discard the supernatant.</li>
		<li>Repeat step 2.3.13.</li>
		<li>Carefully aspirate the remaining S30B supernatant with a 100-200 &#181;L pipette.</li>
		<li>Weigh the wet cell pellet. 
		NOTE: Typical wet cell pellet weight for 1 L of overnight GYM culture (OD600 = 3.0) is ~4.5 g.</li>
		<li>For every 1 g of wet cells, add 0.9 mL of S30B buffer. Resuspend the cells using either a Pasteur pipette or vortex.</li>
		<li>Centrifuge briefly (~10 s) up to 500 &#215; g to sediment the cells. 
		NOTE: The protocol can be paused at this point, and cells can be frozen on either liquid nitrogen or dry ice and stored at -80&#176;C. For safety, wear appropriate personal protective equipment (PPE) when handling liquid nitrogen, including face shields and gloves.</li>
	</ol>
	</li>
</ol>
3. Cell lysis by sonication to obtain the crude cell extract
NOTE: At this stage, the user can choose to disrupt the cells by sonication either in 1 mL fractions (option 1) or as a larger cell suspension (5 mL) in a 50 mL tube (option 2). Both options have been detailed below to ensure reproducibility, as the final volume of the cell suspension can change due to the loss of cells during previous harvesting and wash steps. A new user should attempt option 2.1 first to establish the protocol.
<ol>
	<li>Cell Lysis by sonicating in 1 mL fractions
	<ol>
		<li>Using a 1 mL pipette tip (cut off the end of the tip to increase the bore size), transfer 1 mL of the cell suspension into 2 mL microcentrifuge tubes. 
		NOTE: If the cells are frozen, rapidly thaw the 50 mL tube containing the pellet in lukewarm water prior to cell lysis. Transfer the tube to wet ice as soon as the pellet has begun to defrost, and chill for 10 min.</li>
		<li>Place each microcentrifuge tube in a beaker of ice water, using a plastic tube rack to hold the tube for sonication. 
		NOTE: Due to the sensitivity of the cell extract to overheating, it is critical to ensure that the tubes do not warm up to prevent protein precipitation and reduced enzymatic activity.</li>
		<li>Use a sonicator probe with a 3 mm diameter tip and clean it with 70% (v/v) ethanol and double-distilled water (ddH2O). Lower the sonicator tip into the cell suspension until it is ~1 cm below the liquid surface.</li>
		<li>Input the following settings into the sonicator: 20 kHz frequency, 65% amplitude, 10 s pulse ON time, 10 s pulses OFF time, 1 min total sonication time.</li>
		<li>Run the sonication protocol. Move the tube up/down and sideways during the first two resting cycles to ensure the cells are evenly sonicated. Record the energy input. 
		NOTE: For safety, wear appropriate hearing protection during sonication. The viscosity will decrease as cells are disrupted, and the pale cream wet cell pellet should turn into a homogenous brown fluid. The recommended energy input is 240 J per mL of wet cells. If the cells are only partially lysed, the suspension will still appear cream-colored with viscous clumps of cells, particularly on the sides of the tube.</li>
		<li>Invert the tube 2-3 times and repeat the sonication for an additional one or two 10 s cycles, mixing frequently until the cells are fully disrupted.</li>
	</ol>
	</li>
	<li>Cell Lysis by sonicating a 5 mL cell suspension
	<ol>
		<li>If the cells are frozen, rapidly thaw the 50 mL tube containing the pellet in lukewarm water with shaking before cell lysis. Transfer the tube to wet ice as soon as the pellet has begun to defrost, and chill for 10 min.</li>
		<li>Briefly spin the tube at 500 x g to sediment the cells.</li>
		<li>Place the 50 mL tube in a beaker of ice water for sonication. 
		NOTE: Due to the sensitivity of the cell extract to overheating, it is critical to ensure that the tubes do not warm up to prevent protein precipitation and reduced enzymatic activity.</li>
		<li>Use a sonicator probe with a 6 mm diameter tip and clean it with 70% (v/v) ethanol and ddH2O (see the visual schematic of 6 mm probe in Figure 1). Lower the sonicator tip into the cell suspension (~5 mL) until it is ~1 cm below the liquid surface.</li>
		<li>Input the following settings into the sonicator: 20 kHz frequency, 65% amplitude, 10 s pulse ON time, 10 s pulses OFF time, 1 min total sonication time per mL of wet cells (5 min in total).</li>
		<li>Run the sonication protocol. Move the tube up/down and sideways during the first two resting cycles to ensure the cells are evenly sonicated. 
		NOTE: For safety, wear appropriate hearing protection during sonication. The viscosity will decrease as the cells are disrupted, and the pale cream wet cell pellet should turn into a homogenous brown fluid. Record the energy input. An optimal energy input of 240 J per mL of wet cells (~1200 J in total from 5 min sonication) is recommended.</li>
		<li>If some cells remain intact, follow the guidance from step 3.1.5.</li>
		<li>Transfer the cell extracts into 2 mL microcentrifuge tubes.</li>
	</ol>
	</li>
</ol>
4. Cell extract clarification and run-off reaction
<ol>
	<li>Centrifuge the lysed cells at 16,000 &#215; g for 10 min at 4 &#176;C to remove the cell debris. Transfer the supernatant into 1.5 mL microcentrifuge tubes as 1 mL aliquots.</li>
	<li>Perform the run-off reaction for the cell extracts. Incubate the 1.5 mL tubes containing the cell extracts at 30 &#176;C for 60 min on a heat block or incubator without shaking.</li>
	<li>Centrifuge the cell extracts at 16,000 &#215; g for 10 min at 4 &#176;C. Pool the supernatants into a 15 mL centrifuge tube. Mix the supernatant by inverting the tube five times until homogenous, then keep it on ice. Invert gently to avoid the formation of air bubbles.</li>
	<li>Dilute 10 &#181;L of the cell extract 100-fold with S30B buffer and measure the total protein concentration using a Bradford assay with three technical repeats (see Supplemental Material S2 for Bradford assay guidance).</li>
	<li>If the protein concentration is 20-25 mg/mL, transfer the cell extracts as 100 &#181;L aliquots into new 1.5 mL tubes, flash-freeze in liquid nitrogen, and store at -80 &#176;C. 
	NOTE: For safety, wear appropriate PPE when handling liquid nitrogen, including face shields and gloves.</li>
	<li>If the protein concentration is &#60;20 mg/mL, repeat the crude extract preparation steps to ensure high-quality cell extract and TX-TL yields are comparable to the previously published work5.</li>
</ol>
5. Preparation of plasmid DNA template
<ol>
	<li>Purify the pTU1-A-SP44-mScarlet-I plasmid (pUC19 origin) from a freshly transformed E. coli plasmid strain (DH10&#946;, JM109) grown in 50 mL of LB culture (with 100 mg/mL carbenicillin) using an appropriate plasmid DNA purification kit as per manufacturer&#39;s instructions.</li>
	<li>Elute the plasmid in 2 x 300 &#181;L of nuclease-free water and combine the fractions.</li>
	<li>Add 0.1 volumes (66 &#956;L) of 3 M sodium acetate (pH 5.2).</li>
	<li>Add 0.7 volumes (462 &#956;L) of isopropanol.</li>
	<li>Incubate the DNA at -20 &#176;C for 30 min.</li>
	<li>Centrifuge at 16,000 &#215; g for 30 min at 4 &#176;C and discard the supernatant.</li>
	<li>Add 2 mL of 70% (v/v) ethanol to the DNA pellet.</li>
	<li>Invert the tube 3-4 times to resuspend the plasmid DNA pellet.</li>
	<li>Centrifuge at 16,000 &#215; g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Repeat steps 5.7-5.9 and remove all visible liquid.</li>
	<li>Air-dry the DNA pellet for 10-30 min or dry for 5 min with a vacuum centrifuge.</li>
	<li>Resuspend the dried pellet with 600 &#181;L of nuclease-free ddH2O.</li>
	<li>Measure the DNA concentration and purity using a spectrophotometer.</li>
	<li>Prepare 50-100 &#181;L aliquots and store at -20 &#176;C. 
	&#8203;NOTE: A high DNA concentration in the range of 500-1000 ng/&#181;L is recommended due to the tight volume constraints of cell-free reactions. Dilute the plasmid DNA stock to 80 nM; 168 ng/&#181;L pTU1-A-SP44-mScarlet-I plasmid is equivalent to 80 nM.</li>
</ol>
6. Preparation of the Streptomyces Master Mix (SMM) solution
<ol>
	<li>Amino acid solution
	<ol>
		<li>Use the amino acid sampler kit to avoid manual errors and reduce preparation time, following the manufacturer&#39;s instructions provided online.</li>
		<li>Dilute the 20x amino acid stock solution using ddH2O to a final concentration of 6 mM (5 mM L-Leu).</li>
		<li>Further dilute to 2.4 mM (2 mM L-Leu) within the 2.4x SMM solution (see Table 3). 
		&#8203;NOTE: The final concentration in the TX-TL reaction is 1 mM 19x amino acids and 0.83 mM L-Leu.</li>
	</ol>
	</li>
	<li>Energy solution and additives
	<ol>
		<li>Prepare the other components in the 2.4x SMM solution by following the recipe described in Table 3.</li>
		<li>Alternatively, prepare a 2.4x Minimal Energy Solution (MES), following the recipe described in Table 3.</li>
	</ol>
	</li>
</ol>
7. Setting up a standard S. venezuelae TX-TL reaction
<ol>
	<li>Thaw the cell extract, SMM (or MES) solution, and plasmid DNA on ice. Pre-chill a 384-well plate at -20 &#176;C.</li>
	<li>Set up TX-TL reactions where 25% of the volume is plasmid DNA, 33.33% is cell extract, and 41.67% is SMM solution; keep them on ice to avoid start time bias. 
	NOTE: A standard TX-TL template has been provided (Table 4) to calculate the volume of reagents needed based on the number of reactions. The standard volume for a 33 &#181;L reaction is as follows: 11 &#181;L of cell extract, 13.75 &#181;L of SMM, and 8.25 &#181;L of plasmid DNA.</li>
	<li>Gently vortex the mixture for ~5 s at a low-speed setting to ensure the solution is homogenous. Avoid foaming/bubble formation.</li>
	<li>Transfer 10 &#181;L aliquots into three wells of a 384-well plate as a technical triplicate without introducing air bubbles. Seal the plate with a transparent cover and spin at 400 &#215; g for 5 s.</li>
	<li>Incubate the reaction at 28 &#176;C either in an incubator (for end-point readings) or a plate-reader without shaking. 
	NOTE: Reactions typically require 3-4 h to reach completion. See Supplemental Material S2 for guidance on a plate reader and mScarlet-I standard measurements.</li>
</ol>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>2.5 L UltraYield Flask</td><td>Thomson</td><td>931136-B</td><td></td></tr><tr><td>3-PGA (&gt;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, &gt;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 (&amp;ge;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-EDT&lt;sub&gt;2&lt;/sub&gt; (contact supplier for purity)</td><td>Santa Cruz Biotech</td><td>sc-363644</td><td></td></tr><tr><td>Glucose-6-phosphate (&gt;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 (&gt;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 (&gt;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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 ...

a-high-yield-streptomyces-transcription-translation-toolkit-for

Research

JoVE Journal

Bioengineering

4.0K Views.  South Kensington Campus. Ons protocol biedt een vereenvoudigde workflow voor Streptomyces venezuelae celvrije transcriptie-vertaling. Ons systeem maakt gebruik van de metabole enzymen en routes van dit niet-modelorganisme. De belangrijkste voordelen zijn de heterologe expressie van genen met een hoog GC-gehalte, het verstrekken van een gunstige eiwitdoorstuuromgeving en een open systeem voor het finetunen van biosynthetische routes.Onze methode zal nieuwe mogelijkheden bieden voor het bestuderen van Streptomyc...

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

From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes T&#252;6028

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different conditions often leads to the production of new compounds. Therefore, production cultures of the strains are extracted with ethyl acetate and the crude extracts are analyzed by HPLC. Furthermore, the extracts are tested for their bioactivity by different assays. For structure elucidation the compound of interest is purified by a combination of different chromatography methods.
Genome sequencing coupled with genome mining allows the identification of a natural product biosynthetic gene cluster using different computer programs. To confirm that the correct gene cluster has been identified, gene inactivation experiments have to be performed. The resulting mutants are analyzed for the production of the particular natural product. Once the correct gene cluster has been inactivated, the strain should fail to produce the compound.
The workflow is shown for the antibacterial compound polyketomycin produced by Streptomyces diastatochromogenes T&#252;6028. Around ten years ago, when genome sequencing was still very expensive, the cloning and identification of a gene cluster was a very time-consuming process. Fast genome sequencing combined with genome mining accelerates the trial of cluster identification and opens up new ways to explore biosynthesis and to generate novel natural products by genetic methods. The protocol described in this paper can be assigned to any other compound derived from a Streptomyces strain or another microorganism.

Here we present a detailed protocol of (A) the identification of a natural product with antibiotic activity, (B) the purification of the compound, (C) the first model of its biosynthesis, (D) genome sequencing/-mining and the (E) verification of the biosynthetic gene cluster.

Streptomyces strains are known for their capability to produce a lot of different compounds with various bioactivities. Cultivation under different ...

Genetics

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing biosynthetic pathways. In this manuscript, the use of a highly effective system for the construction of multigene synthetic operons under the control of an inducible T7 RNA polymerase is described. This system allows many genes to be expressed simultaneously from one plasmid. Here, a set of four related vectors, pMGX-A, pMGX-hisA, pMGX-K, and pMGX-hisK, with either the ampicillin or kanamycin resistance selectable marker (A and K) and either possessing or lacking an N-terminal hexahistidine tag (his) are disclosed. Detailed protocols for the construction of synthetic operons using this vector system are provided along with the corresponding data, showing that a pMGX-based system containing five genes can be readily constructed and used to produce all five encoded proteins in Escherichia coli. This system and protocol enables researchers to routinely express complex multi-component modules and pathways in E. coli.

This study describes methods for the T7-mediated co-expression of multiple genes from a single plasmid in Escherichia coli using the pMGX plasmid system.

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

Chemistry

Mass Spectrometry-Guided Genome Mining as a Tool to Uncover Novel Natural Products

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation of their functions in nature and potential human benefits (e.g., for drug discovery applications) are desired. This protocol describes the combination of genome mining (GM) and molecular networking (MN), two contemporary approaches that match gene cluster-encoded annotations in whole genome sequencing with chemical structure signatures from crude metabolic extracts. This is the first step towards the discovery of new natural entities. These concepts, when applied together, are defined here as MS-guided genome mining. In this method, the main components are previously designated (using MN), and structurally related new candidates are associated with genome sequence annotations (using GM). Combining GM and MN is a profitable strategy to target new molecule backbones or harvest metabolic profiles in order to identify analogues from already known compounds.

A mass spectrometry-guided genome mining protocol is established and described here. It is based on genome sequence information and LC-MS/MS analysis and aims to facilitate identification of molecules from complex microbial and plant extracts.

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Immunology and Infection

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Biology

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

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

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