Abstract
Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor transcription rate of Escherichia coli RNA polymerase in this minimal system. We found that addition of transcription elongation factors GreA and GreB to the PURE system increased transcription rates of E. coli RNA polymerase from sigma factor 70 promoters up to 6-fold and enhanced the performance of a genetic network. Furthermore, we reconstituted activation of natural E. coli promoters controlling flagella biosynthesis by the transcriptional activator FlhDC and sigma factor 28. Addition of GreA/GreB to the PURE system allows efficient expression from natural and synthetic E. coli promoters and characterization of their regulation in minimal and defined reaction conditions making the PURE system more broadly applicable to study genetic networks and bottom-up synthetic biology.
Introduction
Diverse biological processes can be reconstituted and studied in vitro using purified proteins or lysates. This approach has facilitated fundamental discoveries in molecular biology and biochemistry, such as DNA replication1 and translation of the genetic code2. The cell-free approach allows construction of systems that would be difficult or impossible to develop in vivo and to perform measurements and experiments that are difficult to conduct in cells. Apart from basic research on biological networks, applications of in vitro systems thus far have include biosensors and molecular synthesis 3–5. E. coli cell-lysate transcription and translation (TX-TL) systems have become popular to engineer and study genetic networks of increasing complexity6–10. Cell-lysate-based TX-TL systems produce high protein yields and allow transcription from native E. coli promoters11. However, lysates have the disadvantage that they contain almost all the proteins and macromolecules present in the cytoplasm at the moment of lysis. For bottom-up synthetic biology and reconstitution studies this is not ideal because the lysate still contains many unknown components and uncontrollable variables.
An appealing transcription and translation system that is commercially available as PURExpress (PURE), is reconstituted from purified components and allows experiments under minimal and defined conditions12. This reconstituted system contains T7 RNA polymerase (RNAP), purified ribosomes, all necessary translation factors from E. coli, tRNAs, enzymes for tRNA aminoacylation and energy regeneration, creatine phosphate as an energy source, and nucleotides and amino acids as precursors. Various genetic networks can be implemented in this system, which to date mostly relied on single subunit phage RNA polymerases for transcription13.
Transcription by the multisubunit E. coli RNA polymerase (EcRNAP) has been reconstituted in the PURE system and can be implemented by either adding the purified holoenzyme to the reaction mix or by co-expressing its subunits14,15. While transcription rates of the EcRNAP in PURE depend on the concentrations of DNA template and EcRNAP, they are generally considerably lower than for phage polymerases. For example, we observed roughly an order of magnitude lower mRNA concentrations synthesized from a consensus sequence sigma factor 70 (σ70) promoter by EcRNAP than by phage T3 RNAP13 under similar conditions. In vivo elongation rates of EcRNAP range between 28 and 89nt/s16 and are comparable to the values reported for T7 RNAP in vitro.17,18 Transcription elongation factors affect EcRNAP transcription elongation rate either by sensitizing or suppressing RNAP pausing. Elongating RNAP frequently backtracks along the DNA forming a transcriptionally inactive state. Transcription elongation factors GreA and GreB bind to backtracked RNAP and catalyze the endonucleolytic cleavage of nascent RNA within the RNAP active site allowing transcription to continue19. GreA resolves smaller backtracking events by cleaving 2-3 nt from the 3’end of the RNA, whereas GreB can also rescue longer backtracked complexes20. GreA and GreB are known to increase transcription elongation rate and stimulate promoter escape in a subset of promoters19–22, but GreA and GreB transcription elongation factors are not present in the PURE system.
Here we show that E. coli transcription elongation factors GreA and GreB enhance EcRNAP transcription rates in the PURE system up to 6-fold to reach the rate of T7 RNAP transcription in the system. We go on to show that an increase in transcription rates can be observed for several different synthetic and natural σ70 E. coli promoters. Furthermore, we used the enhanced system to study natural E.coli promoters involved in flagella biosynthesis and their activation by two different transcriptional activators in vitro, under defined conditions.
Results and discussion
EcRNAP can be added to the PURE system to allow transcription of DNA templates carrying E. coli promoters14,15 but mRNA synthesis and subsequent protein production is more efficient using phage polymerases such as T7 or T3 RNAP13. In bacterial cells multiple proteins can increase RNAP activity, which are not present in the minimal PURE system. The transcription elongation factors GreA and GreB from E. coli increase overall transcription elongation rates and stimulate promoter escape in a subset of promoters by re-activating backtracked elongation complexes19–22. We added transcription elongation factors GreA and GreB to a PURE reaction containing EcRNAP with σ70 (holoenzyme) and a DNA template expressing EGFP under control of a consensus sequence E. coli σ70 promoter. We measured concentrations of full-length mRNA using fluorescent FRET probes that bind to a target region at the 3’ end of the mRNA23 (Fig. 1A) and found that in the presence of GreA and GreB mRNA concentrations increased faster and to higher concentrations than without the elongation factors (Figure 1). The transcription rate increase mediated by GreA and GreB followed hyperbolic kinetics and plateaued at concentrations above 5 μM for both GreA and GreB. At the plateau GreA and GreB increased transcription rates about 3-fold and final EGFP protein synthesized about 2-fold for the DNA template concentration tested (Fig. 1B, C). When GreA and GreB were added in combination, we did not observe a significant synergistic effect on mRNA or protein synthesis (Supplementary Fig. 1). We nonetheless used both proteins in an enhanced PURE (ePURE) reaction containing 10 μM of GreA and 10 μM of GreB in addition to the E. coli RNAP holoenzyme (0.2 μM EcRNAP, 1 μM σ70) for all subsequent experiments. Apart from these protein additions we did not further modify the commercial PURE system (see Materials and Methods). During a TX-TL reaction in ePURE we observed up to 10-fold higher mRNA concentrations, which also translated to an increased EGFP synthesis (Fig. 1D, E).
By testing various DNA template concentrations we observed an up to 6-fold increase of transcription rate in ePURE compared to a PURE reaction supplemented with E. coli RNAP holoenzyme alone (Fig. 2A). Increased mRNA synthesis led to 3-fold higher final EGFP levels, and the advantage of using the ePURE reaction was strongest for lower DNA template concentrations (Fig. 2B). In the ePURE system we observed comparable mRNA and EGFP synthesis from an E. coli σ70 promoter and a T7 RNAP promoter (Fig. 2). The ePURE improvement should thus facilitate using E. coli RNAP for transcription under defined conditions.
We next asked whether GreA and GreB can improve transcriptional elongation rates for genes other than EGFP. In order to test this, we measured synthesis of full-length mRNA from two additional genes: asr (309 bp), and chiP (1407 bp) which are roughly half and twice as long as the EGFP gene (717 bp), respectively. Transcription rates of both genes were increased in the ePURE system in the presence of GreA and GreB (Supplementary Fig. 2). In the non-optimized PURE system transcription rates were below the detection limit for the longer gene and increased to measurable levels in the presence of GreA and GreB. The transcription rate of the shorter gene increased 8-fold (Supplementary Fig. 2).
In order to determine if the ePURE system also increases the transcription rate for promoters other than the strong σ70tet promoter we characterized in Figures 1 and 2, we tested the system on 16 additional synthetic and natural σ70 promoters (Fig. 3). The panel was composed of nine constitutive promoters from the registry of standard biological parts (http://parts.igem.org), the BBa_J231xx-series, which are well-characterized in vivo and in vitro,24–26, two constitutive promoters proC and proD 27, several natural repressible promoters (promoter of the trp operon, lac UV5 promoter, the phage λPR promoter), and three synthetic repressible promoters (Ptet 28, and the σ70 consensus sequence promoters Pσ70lac and Pσ70tet 13). In the ePURE system EGFP synthesis increased for 14 of the 17 promoters we characterized (Fig. 3A). Our results on the constitutive J231xx-promoters compare well with relative promoter strengths measured in lysate-based TX-TL reactions25,26. We found that the presence of GreA and GreB in the ePURE system enhanced expression particularly for strong promoters. These results suggest that at least for strong promoters transcriptional pausing during elongation severely limited transcription by EcRNAP in the PURE system. However, we cannot rule out effects on initiation of transcription as it has been shown that GreA and GreB can influence promoter escape differently for different promoters22. By increasing the range of synthesis rates that can be attained with E. coli promoters the ePURE system will be useful in implementing and characterizing genetic networks based on native E. coli promoters.
As an example of this we assembled a simple synthetic 2-gene cascade that consisted of T7 RNAP transcribing the rpoD gene to synthesize σ70, which then, together with EcRNAP core enzyme, activated EGFP expression (Fig. 3B). In order to study the effect of DNA template concentration on the performance of the network, we additionally analyzed the circuit at a low and a high DNA template concentration of 10 and 30 nM, respectively (Fig. 2B). The PURE system failed to run the genetic cascade at either DNA concentration and yielded output comparable to the negative control lacking the rpoD template. The ePURE system on the other hand yielded increased expression of the reporter, which was further boosted at higher DNA concentrations. This demonstrates that addition of GreA and GreB enables the implementation of genetic networks involving EcRNAP. In fact, in this case ePURE was necessary for the cascade to function, and simply increasing DNA template concentrations was not sufficient to improve performance of the network in the PURE system.
We next tested whether the ePURE system would allow us to study an endogenous bacterial genetic network and chose to analyze transcriptional regulation of native E. coli flagellar promoters. Two main regulators, the FlhDC transcriptional activator and FliA, the flagellar sigma factor, σ28, are thought to activate the genes in a tightly controlled temporal order29,30. FlhDC is known as the master regulator and activates σ70-dependent transcription from class 2 promoters. One of the genes FlhDC activates is fliA coding for σ28, which then activates itself and other genes in a positive autoregulation31. Many of the more than 50 genes in the flagellar regulon, which are divided into at least 17 operons, are transcribed from multiple promoters, and can be activated by both FlhDC and σ2829,32. Their regulation has been studied extensively in vivo using genetics and promoter fusions29,30,32. In a complementary approach, this complex regulatory system can also be studied outside of cells, under reaction conditions that eliminate unknown factors. Activation of several flagellar class 2 and class 3 promoters by FlhDC and σ28 was shown by in vitro transcription experiments31,33,34 and binding of the FlhDC activator to putative promoters was demonstrated by in vitro binding assays35.
We analyzed eight flagellar promoters coupled to an EGFP reporter in the ePURE system and show their activation by FlhDC and σ28 in the defined TX-TL system (Fig. 4A). We based our expectations on the EcoCyc E. coli database36, which contains information on experimentally known and bioinformatically predicted transcriptional activation. To synthesize our DNA templates we fused 150 to 250 bp long promoter regions that contained the annotated σ70 and σ28 promoters as well as FlhDC binding sites to identical, strong ribosomal binding sites followed by the EGFP reporter gene. To test their activation, we separately pre-synthesized the FlhDC and the σ28 activators and then added these to an ePURE reaction containing GreA, GreB, EcRNAP, and a DNA template with the respective flagellar promoters.
All eight promoters tested showed no detectable activity in the absence of FlhDC and σ28. When the reaction contained either of the two activators, we observed the expected activation pattern with widely differing promoter strengths (Fig. 4A). Both activators in combination generally did not improve expression compared to only one activator. Most of the time the presence of both activators even led to decreased expression. We attribute this effect to competition between both activators for binding to DNA31 and to the RNAP core enzyme, which binds σ28 with a higher affinity than σ7034. We hypothesize that concentrations of EcRNAP and activators might be different in cells than in our in vitro assay, which could explain why the promoters did not show additive activation as observed in vivo32. When considering activation by a single activator, two out of eight promoters, fliE and flgK, deviated from the annotated regulation pattern in EcoCyc. The fliE promoter was predicted to be activated by both σ2837 and FlhDC35 but we only detected low activation by FlhDC. For the flgK promoter only activation by σ28 was annotated on EcoCyc and previously shown34. We observed strong activation by σ28 but also a low but significant activation by FlhDC. A computational search for FlhDC did not identify a FlhDC binding site upstream of the flgK gene in E. coli.35 In Salmonella typhimurium, however, transcription of flgK is activated by both σ70/FlhDC and σ2838. Additionally, our study provides experimental evidence for activation of several promoters by FlhDC and σ28, which have previously only been predicted computationally, such as activation of flgB, fliE and fliD by FlhDC35 and flgM by σ2839. Thus, our analysis of flagellar promoters in defined conditions demonstrates that native gene activation mechanisms can be studied using the ePURE system. The finding that FlhDC is a strong transcriptional activator for a number of different promoters should furthermore be useful for the assembly of synthetic genetic networks.
Using the genes for FlhDC and σ28 and two promoters that showed activation by both activators, we built a synthetic gene network (Fig. 4B), which was implemented and characterized in a microfluidic nano-reactor device13. T7 RNAP transcribes the genes coding for FlhD and FlhC. FlhDC then activates σ70 E. coli RNAP to express the Citrine reporter, which we placed under control of the flgK promoter. A control reaction, which did not contain the FlhD and FlhC DNA templates, again demonstrated activation of the flgK promoter by the FlhDC activator (Fig. 4C). Transiently we added DNA templates carrying the fliA gene (σ28) and a Cerulean reporter gene, both under control of the fliA promoter, which leads to positive autoregulation of σ28. σ28 further activates both reporters leading to a fluorescence increase of Citrine and Cerulean in the presence of the fliA and Cerulean templates (Fig. 4C). This 5-gene network demonstrates that complex genetic networks dependent on the E. coli RNAP and native E. coli promoters can be assembled in the ePURE system.
E. coli promoters offer a wide and well-characterized repertoire of promoter-regulator pairs and E. coli promoters are also highly modular40 making transcription by E. coli RNAP interesting for in vitro synthetic biology11. While lysate-based TX-TL systems can be prepared with high activities of the EcRNAP11,41, we have found activity of the EcRNAP in the reconstituted PURE system to be too low for many applications. PURE is a minimal system that can be rationally improved by a bottom-up approach. Here we enhanced transcription in the PURE system by adding purified transcription elongation factors GreA and GreB, which indicates that transcriptional elongation was limiting performance of EcRNAP in the PURE system. Addition of GreA and GreB increased EcRNAP transcription rates from a strong σ70 promoter to levels observed with phage T7 RNAP and significantly increased transcription from a number of synthetic E. coli promoters of different strengths. We used the ePURE system to characterize activation of native E. coli flagellar promoters by the transcriptional activators FlhDC and σ28, demonstrating that the ePURE system is useful for the characterization of genetic regulation under defined conditions. Inclusion of GreA and GreB proteins should furthermore be useful to increase protein yields in the PURE system when it is desirable to use E. coli promoters.
Materials and Methods
DNA template preparation
Linear DNA templates were produced by two-step PCR as described previously13,23 using the primers listed in Table S1. Flagella promoters were PCR amplified from E. coli BL21(DE3) genomic DNA and replaced the 5’extension primer during two-step PCR. All linear DNA templates prepared for this study are listed in Table S2.
Preparation of GreA, GreB and EcRNAP holoenzyme
EcRNAP subunits (expression plasmid pVS10) were co-expressed in E. coli Xjb(DE3) cells (Zymo Research, Irvine, CA, USA) and the α2ββ′ω assembly (β′ subunit contained C-terminal His6-tag) was purified by a combination of immobilized-metal affinity, heparin and anion exchange chromatographies as described42. E. coli σ70 protein containing N-terminal His6-tag (expression plasmid pET28-σ7043) was expressed in E. coli Xjb(DE3) cells and purified by a combination of immobilized-metal affinity, heparin and anion exchange chromatographies as described for EcRNAP except that the lysis and the wash buffers during immobilized-metal affinity chromatography contained 1 M NaCl. E. coli GreA and GreB proteins containing C-terminal His6-tags (expression plasmids pIA578 and pIA577, respectively) were purified by immobilized-metal affinity chromatography followed by gel filtration as described44. All proteins were dialyzed against the storage buffer (20 mM Tris-HCl pH 7.9, 150 mM NaCl (1M NaCl for GreA and GreB), 0.1 mM EDTA, 50% glycerol, 0.1 mM DTT) and stored at -20°C. A stock solution of 50x holoenzyme (1:5 E. coli core RNAP : σ70 factor at 10 μM and 50 μM) was prepared by incubating the proteins in storage buffer at 30°C for 20 min, then the stock was stored at -20°C until use.
Batch reaction setup and measurements
TX-TL reactions were performed in the PURExpress In Vitro Protein Synthesis kit (New England Biolabs) supplemented with Protector RNase Inhibitor (Roche), 1 μM Cy3 and Cy5 binary probes23, 200 nM E. coli core RNAP and 1 μM σ70 factor. The ePURE system additionally contained 10 μM of each GreA and GreB transcription elongation factors. Platereader batch TX-TL reactions, and measurement of the mRNA concentration were performed as previously described23. The initial mRNA synthesis, or transcription rate (TX), was determined by fitting the mRNA concentration (m) of the first 40 min of the reaction to: where t is time and deg signifies the mRNA degradation rate (fixed at 0.0085 min−1)23. The final EGFP concentration was determined at the plateau of the protein synthesis reaction.
To test activation of flagellar promoters by FlhDC and σ28, we pre-synthesized the activators from T7 RNAP templates in a standard PURE reaction without EcRNAP and Gre proteins. FlhDC was produced by combining flhD and flhC templates at 10nM each, and σ28 was produced from 10 nM fliA template for 100 min at 37°C. The activators then were stored in aliquots at -80°C until use. These were prepared by combining the FlhDC and σ28 presynthesis reactions 1:1 for testing activation by both activators and by diluting 1:1 with Tris buffer for testing of each activator separately.
Flagellar gene network in a nano-reactor device
We assembled the genetic network from 5 individual DNA templates. Final concentrations were 1 nM for PT7-flhD, 2.5 nM for PflgK-Citrine, and PT7-flhC, and 2 nM for PfliA-fliA and PfliA-Cerulean. The templates PfliA-fliA and PfliA-Cerulean were only added transiently. The microfluidic chip was prepared and used as described13 using a dilution time, td, of 39.6 ±0.4 min. Citrine and Cerulean concentrations were determined from a calibration with purified proteins10.
Supporting Information
Supporting figures S1 and S1. Supporting tables S1 and S2.
Author Information
Corresponding author: E-mail Sebastian.maerkl{at}epfl.ch
Author contributions: LLM and HN contributed equally to this work
Acknowledgements
The authors would like to thank Irina Artsimovitch (Ohio State University) for providing expression plasmids for EcRNAP, GreA and GreB.