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Letter
Designing synthetic flexible gene regulation networks using RNA devices in cyanobacteria Akiyoshi Higo, Atsuko Isu, Yuki Fukaya, and Toru Hisabori ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00201 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016
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Designing synthetic flexible gene regulation networks using RNA
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devices in cyanobacteria
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Akiyoshi Higoa;b, Atsuko Isua;b, Yuki Fukayaa;b, and Toru Hisaboria;b #
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a
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Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan; bCore Research for
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Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST),
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Tokyo 102-0075, Japan
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of
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#
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Research, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-Ku, Yokohama
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226-8503, Japan; Tel. 81-45-924-5234; Fax. 81-45-924-5268, email:
[email protected] Corresponding author: Laboratory for Chemistry and Life Science, Institute of Innovative
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ABSTRACT
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In recent years, studies on the development of gene regulation tools in cyanobacteria have been
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extensively conducted towards efficient production of valuable chemicals. However, there is
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considerable scope for improving the economic feasibility of production. To improve a recently
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reported gene induction system using anhydrotetracycline (aTc)–TetR and an endogenous gene
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repression system using small antisense RNA in the filamentous nitrogen-fixing
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cyanobacterium Anabaena sp. PCC 7120 (Anabaena), we constructed a positive feedback loop,
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in which gfp and a small antisense RNA for tetR are controlled by an aTc-inducible promoter.
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GFP expression in this improved system was higher and longer than the system lacking tetR
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repression. In addition, using TetR aptamer and a riboswitch, we succeeded in achieving a
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superior and longer induction of GFP expression even under high-light conditions. Hence,
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efficient gene induction systems were established in Anabaena by designing a gene regulation
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network using RNA-based tools.
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Key words: cyanobacteria, gene regulation system, small RNA, riboswitch
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INTRODUCTION
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By designing artificial genetic circuits, in which regulatory devices are appropriately combined,
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various types of systems have been created to perform specific tasks in living cells since the
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first construction of the genetic oscillator1 and toggle switch.2 Inspired by electrical engineering
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and computer science, researchers working in the field of synthetic biology have produced
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biological counters,3 Boolean logic gates,4 band-path filters,5 and memory.6 In addition, such
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synthetic biology approach has been applied to the development of biosensors and production of
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valuable chemicals7
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Although cyanobacteria have been expected to play a role in the production of useful
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substances and biofuels because of their photosynthetic ability,8 lack of genetic devices for
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efficient gene regulation in these organisms has limited their productivity.9, 10 To overcome this
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challenge, studies on development of gene regulation tools have been actively conducted in
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recent years.8 Among them, gene induction system consisting of an inducer, anhydrotetracycline
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(aTc), and a transcriptional repressor, TetR, showed a wide dynamic range in some
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cyanobacteria, although photolability of aTc limited the usefulness of this system.11–13 A
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theophylline riboswitch-based gene induction system has also been successfully used in
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cyanobacteria.14, 15 In addition, gene repression systems have been developed in cyanobacteria.
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A small RNA based on an Escherichia coli IS10 RNA-IN/OUT system was used for the
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repression of exogenous gfp expression.12 Furthermore, we and the other group succeeded in
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controlling endogenous gene expression by using artificial small antisense RNA (asRNA)13 and
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CRISPRi system,16 respectively. However, the number of studies on construction of a genetic
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network using such gene regulation devices is limited. Recently, Immethun et al. built a
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two-input AND gate in the unicellular cyanobacterium Synechocystis sp. PCC 6803,17
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demonstrating the potential of synthetic biology in cyanobacteria.
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For the industrial production of valuable materials in cyanobacteria, it is important to
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consider the cost of production. Because synthetic inducers are generally expensive, the amount
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of these inducers used to induce gene expression should be minimal to reduce production costs.
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In addition, photolability-related issues of aTc should be solved when TetR system is used in
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photosynthetic cyanobacteria. Hence, we improved our previous TetR system in Anabaena13
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using a synthetic biology approach. We designed a positive feedback loop and succeeded in
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achieving greater and prolonged gene induction using only a small amount of aTc. Furthermore,
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by combining a TetR aptamer that inactivates TetR repression activity and a riboswitch that
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terminates transcription in the presence of adenine, we enabled strong and prolonged gene
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induction under high-light conditions.
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RESULTS AND DISCUSSION
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In our previous study, we demonstrated that the amount of TetR expressed in cells is a
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determinant of responsiveness to the inducer aTc.13 Thus, tetR was expressed under the control
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of nirA promoter, which is active in the presence of nitrate and is repressed in the absence of a
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nitrogen source.18 Therefore, gene expression is induced under nitrogen-fixing conditions13
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(pCA004 in Figure 1 and supplemental Table S1). In the pCA004 system, induction by nitrogen
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starvation as well as by aTc was possible. Lowering the TetR amount by the promoter
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engineering of PnirA facilitated the creation of a strong induction system (pCA00513), which,
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however, had a narrow dynamic range. In this study, we designed a positive feedback loop to
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induce gene expression strongly with a wide dynamic range. We combined two systems, TetR
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induction and small asRNA repression, which were previously developed by us.13 A small
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asRNA was designed to repress tetR expression, and the small asRNA and the reporter gene gfp
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were expressed under the control of PL03, which is regulated by aTc and TetR (pCA011 in
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Figure 1 and supplemental Table S1). In the case of the pCA011 system, once these genes are
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induced, the amount of TetR in the cell should severely decrease and consequently significant
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gene induction should occur. Here, we expressed TetR with a protease tag LVA13 at the
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C-terminus for the efficient turnover of this protein in the cell (Figure 1).
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We introduced two plasmids, pCA004 that lacked the asRNA gene for tetR repression
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and pCA011, into Anabaena. We then measured GFP fluorescence in these two strains, which
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was induced by indicated concentrations of aTc for 24 h in nitrate-replete (+N) medium (Figure
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2). In addition, we measured GFP fluorescence induced by nitrogen starvation (−N) for 72 h as
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well. GFP fluorescence was very low without the inducer in +N conditions in each strain
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(Figure 2, #1 and #2). As expected, when GFP expression was induced by aTc or nitrogen
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starvation, the asRNA-expressing strain showed stronger fluorescence than the control strain.
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However, even in the presence of 2,000 ng/ml aTc, the pCA011-contaning strain showed only
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one-fourth of GFP fluorescence in a strain without tetR (pCA00113), indicating that a further
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improvement is possible. Western blotting analysis confirmed that the amount of TetR
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decreased in response to aTc in the asRNA-expressing strain but not in the control strain (Figure
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3A). This result demonstrates the plasticity of small asRNA, which can repress a gene in an
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artificial network as well as an endogenous gene.13 When 20 ng/mL aTc was added, the TetR
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amount in the asRNA-expressing strain decreased within 24 h. Even after 72 h, the amount of
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TetR was lower in the asRNA-expressing strain than in the control strain (Figure 3B). In
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addition, GFP expression continued for 72 h in the former strain, while it gradually decreased
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after 24 h in the latter strain (Figure 3B). These results indicate that construction of an artificial
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gene regulatory network complemented photolability of aTc and that such a synthetic biology
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approach is useful in Anabaena.
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Next, we wanted to improve the system in which tetR is expressed under the control of
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PpetE, which would be active irrespective of nitrogen source.19 We designed a similar positive
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feedback loop, in which tetR expression under PpetE is repressed by a small asRNA (pCA012 in
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Figure 1 and supplemental Table S1). A control plasmid pCA002 and pCA012 were introduced
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into Anabaena. We confirmed that the amount of TetR decreased in response to aTc in the
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pCA012-harboring strain (Supplemental Figure S1A). Furthermore, GFP fluorescence in the
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strain was higher than that in the control strain (Supplemental Figure S1B). These results
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suggest that the small asRNA approach has a potential to improve a variety of gene expression
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systems.
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We then constructed a positive feedback loop, in which the amount of TetR is not
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affected but the TetR repressor function is inhibited by TetR-inducing peptide (TiP)20 or TetR
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aptamer.21, 22 A similar design for a prolonged induction had been previously proposed.11 TiP is
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generally used with scaffold proteins such as TrxA from E. coli.20 In this study, we used E. coli
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TrxASS, in which two cysteine residues at the active site were substituted with serine to avoid
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undesirable thiol-disulfide exchange reactions with endogenous proteins in Anabaena.
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Unexpectedly, compared with the control strain, each Anabaena strain containing
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Tip-TrxASS-expressing plasmid pCA013 or TetR aptamer-expressing plasmid pCA014 showed
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no significant differences in GFP fluorescence at various concentrations of aTc (Figure 2, #1, #3,
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and #4). An LVA tag at the C-terminus of TetR or rapid turnover of TetR may interfere with
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stable interaction between TetR and TiP-TrxASS or the TetR aptamer. Next, we expressed the
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TetR aptamer with a tRNA scaffold23 (pCA015 plasmid in Figure 1 and supplemental Table S1)
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to stabilize the TetR aptamer in the cells. While GFP fluorescence in the pCA015-containing
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strain was 5.5-fold higher than that in the control strain under non-induced conditions, the
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former strain exhibited a 9-fold higher GFP fluorescence when induced under nitrate depletion
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than that exhibited by the control strain (Figure 2, #1 and #5). In addition, GFP fluorescence in
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the pCA015-containing strain was higher by induction with, in particular, a small amount of aTc
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(20 ng/mL) than the fluorescence in the control strain. These results indicate that a strong GFP
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induction by aTc or nitrate depletion was achieved, while the pCA015 system was slightly leaky
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compared to the control system.
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Time-course analysis of GFP induction was performed in a tRNA-scaffold TetR
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aptamer-expressing strain using pCA015. In this strain, GFP was strongly expressed even after
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72 h of induction with 20 ng/mL aTc as opposed to the control strain (Figure 4A). When nitrate
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was depleted from the culture medium in both strains, the amount of TetR clearly decreased
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after 8 h (Figure 4B). Unexpectedly, GFP gradually accumulated after 24 h of nitrate depletion
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in these two strains (Figure 4B), in contrast to when GFP was induced by aTc (Figure 4A).
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Development of rapid induction by decrease of TetR amount is required in the future.
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However, such a gradual induction may be preferred in some cases because rapid induction of
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some genes and successful modulation of metabolism immediately after nitrate depletion may
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inhibit the complex heterocyst differentiation process and nitrogen fixation.
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For constructing a flexible gene induction system, we adopted another approach: a
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riboswitch was used to control the expression of tetR. Riboswitch xpt(C74U)/metE,24 an off
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switch that terminates transcription in the presence of adenine, was inserted downstream of PnirA
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in pCA015. The resultant plasmid pCA016 (Figure 1 and supplemental Table S1) was
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introduced into Anabaena. In the pCA16-containing strain, addition of adenine decreased the
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amount of expressed TetR in a concentration-dependent manner (Figure 5A). This result clearly
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indicates that the riboswitch is functional in Anabaena. The amount of TetR after 24 h
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incubation with 250 µM adenine was one-tenth of that in the absence of adenine (Figure 5A).
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Next, GFP was induced by aTc or nitrate depletion both in the presence and absence of adenine.
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After cells were incubated in the presence or absence of 250 µM adenine for 48 h under +N
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conditions, aTc was added to the culture medium and GFP expression was induced for 24 h.
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Furthermore, GFP fluorescence was induced by incubation for 72 h under −N conditions
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irrespective of the presence of adenine. When adenine was not supplied, GFP expression was
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induced by aTc in a concentration-dependent manner (Figure 5B). Adenine alone induced GFP
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fluorescence, and aTc further induced the fluorescence in +N conditions. In addition, adenine
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alone strongly induced GFP under −N conditions. These results indicate that flexible gene
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induction is possible in the pCA016 system by the addition of adequate quantities of two
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inducers, aTc and adenine, and by the selection of specific growth conditions. Supplemental Figure S2 shows microphotographs of the pCA016 strain. Adenine alone
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induced GFP fluorescence sparsely but strongly across the filaments. GFP fluorescence of the
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majority of cells was strongly induced by aTc alone or both of aTc and adenine. These results
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suggest that the pCA016 induction system exhibits all-or-none response rather than a graded
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one.
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When aTc is used as an inducer in gene induction systems, photolability of this molecule
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may be problematic, especially under high-light conditions. Indeed, 20 ng/mL aTc hardly
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induced GFP expression in the pCA015-harboring strain under high-light (150 µmol photons
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m−2 s−1) conditions as opposed to the results under low-light (30 µmol photons m−2 s−1)
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conditions (Figure 6A). Addition of 200 ng/mL aTc induced GFP expression for a short period
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(24 h) in the strain under high-light conditions but at a lesser extent than that induced under
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low-light conditions with addition of 20 ng/mL aTc. To overcome these photolability-related
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problems observed in aTc, we examined the adjusted induction conditions by decreasing the
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amount of TetR via addition of adenine in the pCA016-containing strain. Addition of only 200
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ng/mL aTc hardly induced GFP expression under high-light conditions (Figure 6B, #1). In
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contrast, addition of 250 µM adenine slightly induced GFP after 72 h under high-light
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conditions in this strain (Figure 6B, #2). When both aTc and adenine were added, GFP
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expression was induced after 24 h and its expression continued until 72 h (Figure 6B, #3). When
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adenine was added 24 h before the addition of aTc and irradiation with high light intensities, 20
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or 200 ng/mL aTc induced GFP expression to a greater extent (Figure 6B, #4, and #5) than that
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induced when aTc and adenine were added simultaneously.
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The results shown in Figure 6B indicate that construction of an artificial gene regulatory
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network enabled gene induction with a wide dynamic range in the aTc-TetR system even under
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high-light conditions. The theophylline riboswitch system14, 15 may be alternatively used for
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gene induction under high-light conditions because theophylline is insensitive to light. However,
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the theophylline riboswitch system shows a narrow dynamic range compared with the aTc-TetR
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system (Supplemental Figure S3). In addition, a functional RNA gene could not be induced by
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the theophylline riboswitch system, in which translation is activated in the presence of
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theophylline.14, 15
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In the present study, we demonstrated that purpose-dependent gene regulatory networks
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could be created by simple combination of gene regulation components, TetR and RNA-based
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devices. Complex gene regulation networks consisting of non-coding RNAs and proteins occur
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ubiquitously in nature. For example, similar to the TetR aptamer that sequesters TetR,22 a small
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non-coding RNA, whose function is controlled by a riboswitch, sequesters a regulator protein to
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prevent expression of ethanolamine utilization genes in Firmicutes. 25, 26 Another example was
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recently demonstrated in the unicellular cyanobacterium Synechocystis sp. PCC 6803: a
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feed-forward loop consisting of a transcriptional regulator and a small non-coding RNA that
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prevents translation of target mRNAs is essential for rapid acclimation response to high-light
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stress.27 In addition, development of RNA-based gene regulation tools has been actively pursued
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in cyanobacteria over the past few years12–16, 28, 29 because of their ease of design.30 Inspiration
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from natural systems, as well as electrical engineering and computer science, should make it
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possible to create various types of gene regulation systems, in which genes of interest are
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robustly controlled even under outdoor conditions, by using especially RNA-based devices.
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Construction of such gene regulation networks would help to generate valuable products using
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cyanobacteria in the future.
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METHODS
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Bacterial strains and growth conditions
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Anabaena strains were routinely grown at 30°C at 30–35 µmol photons m−2 s−1 in BG11
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medium31 supplemented with 20 mM HEPES-NaOH (pH 7.5) and 5 µg/mL neomycin sulfate
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unless otherwise stated. For nitrogen starvation experiments, cells were grown in the same
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medium lacking NaNO3 (BG110). Liquid culture was bubbled with air containing 1.0% (v/v)
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CO2.
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Plasmid construction
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DNA fragments were inserted between the EcoRI and BamHI sites of pRL25c,32 a shuttle vector
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replicating in Anabaena (Figure 1). Detailed sequences are described in the Supplemental
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Information.
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Measurement of GFP fluorescence
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GFP fluorescence from Anabaena cultures was measured as described previously.13 Aliquots (3
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ml) of log-phase cultures (OD750 = 0.1–0.2, 48 h after inoculation) of GFP-expressing Anabaena
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strains were moved into 12-well plates with the indicated amount of aTc or with theophylline
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and 10 mM bicarbonate and then incubated for 24 h at 30–35 µmol photons m−2 s−1. Then,
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fluorescence intensities of GFP expressed in Anabaena cultures (OD750 = 0.2–0.4) were
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measured using a fluorescence spectrophotometer FP-8500 (JASCO, Tokyo, Japan) after the
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optical density of Anabaena at 750 nm (OD750) was adjusted to 0.1–0.12 with BG11 or BG110
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medium supplemented with 20 mM HEPES-NaOH (pH 7.5). An excitation wavelength of 488
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nm and an emission wavelength of 510 nm were used. To obtain the expression level in the
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GFP-expressing Anabaena strains, the fluorescence intensity of the cells containing the empty
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vector was subtracted as the background.
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Total protein extraction
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Cultures of Anabaena strains were centrifuged at 4°C at 2,000 g for 3 min. The cell pellets were
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frozen in liquid nitrogen and stored at −80°C until use. The cells were re-suspended in SDS
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sample buffer [125 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 6% (v/v)
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2-mercaptoethanol, and 0.01% (w/v) bromophenol blue], and immediately boiled for 5 min.
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After centrifugation at 20,000 g for 10 min, the supernatants were collected as total protein.
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Protein concentration was determined with a CB-X protein assay kit (G-Biosciences, St. Louis,
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MO, USA) using BSA as a standard.
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Western blotting analysis
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Equal amounts of total protein (5 µg and 200 ng for detection of TetR and GFP, respectively)
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obtained from various cell lysates were separated on a denaturing SDS-PAGE gel and blotted
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onto the PVDF membrane. TetR and GFP were detected using a TetR monoclonal antibody
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(Takara Bio, Kusatsu, Japan) and a GFP polyclonal antibody (Sigma-Aldrich, St. Louis, MO,
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USA), respectively, with a chemiluminescent kit (ImmunoStar LD, Wako, Osaka, Japan).
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Fluorescence microscopy
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Fluorescence images were taken on a fluorescence microscopy (model IX73, Olympus, Tokyo,
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Japan) with a mirror unit U-FBNA and U-FGW, for observation of GFP fluorescence and
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phycobiliprotein autofluorescence, respectively. Cultures were bubbled with air containing
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1.0% (v/v) CO2.
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Supporting Information
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A document describing the DNA sequences used in this study, as well as summary of aTc-TetR
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induction systems used in this study in Table S1 and Figure S1-S3.
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Abbreviations: asRNA, antisense RNA; aTc, anhydrotetracycline; GFP, green fluorescent
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protein; TiP, TetR-inducing peptide.
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Acknowledgments
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We thank the Biomaterial Analysis Center at the Tokyo Institute of Technology for helping us
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with DNA sequencing technique. This work was supported by the Core Research of Evolutional
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Science and Technology program (CREST) at the Japan Science and Technology Agency (JST).
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Author Contributions
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AH performed the experiments and data analysis. AI, YF, and TH assisted with the project. AH
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prepared the initial draft of the manuscript, and AH and TH edited the manuscript.
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Notes
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The authors declare no competing financial interest.
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Figure legends
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Figure 1. The basic design of plasmids used in this study.
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Figure 2. Induction of gfp expression by aTc or nitrate depletion through synthetic gene
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regulation networks. Anabaena cells were treated with different concentrations of aTc for 24 h
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under nitrate-replete conditions (+N) or were grown under nitrogen starvation conditions (−N)
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for 72 h; GFP fluorescence was then measured and normalized (optical density at 750 nm). The
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experiments using each plasmid were labeled #1 to #5 in the figure. Data represent the mean ±
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SD (n = 3 from independent cultures).
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Figure 3. Repression of TetR by small asRNA. (A) Dependence of TetR amount on aTc
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concentrations. Different concentrations of aTc were added to the medium. After 24 h, proteins
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from each strain grown under +N conditions were extracted, and TetR was detected using a
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monoclonal anti-TetR antibody by western blotting. (B) Time-course analysis of TetR and GFP
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amount after the addition of 20 ng/mL aTc. Total proteins from each strain grown under +N
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conditions were extracted after the indicated time and analyzed by western blotting.
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Figure 4. Induction of GFP in a TetR aptamer-expressing strain. (A) Time-course analysis of
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GFP amount after the addition of 20 ng/mL aTc when the strain was grown under +N conditions.
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Total proteins from each strain were extracted after the indicated time and analyzed by western
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blotting. (B) Time-course analysis of TetR and GFP amount after nitrogen starvation. Total
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proteins from each strain were extracted after the indicated time and analyzed by western
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blotting.
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Figure 5. Induction of GFP by aTc and adenine with an adenine off riboswitch. (A) The amount
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of TetR in strains harboring pCA015 or pCA016 with different concentrations of adenine under
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+N conditions. Western blotting analysis was performed using a monoclonal anti-TetR antibody.
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(B) Anabaena strain harboring pCA016 was treated with different concentrations of aTc for 24
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h under nitrate-replete conditions (+N) or was grown under nitrogen starvation conditions (−N)
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for 72 h in the absence or presence of 250 µM adenine. GFP fluorescence was then measured
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and normalized (optical density at 750 nm). Data represent the mean ± SD (n = 3 from
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independent cultures).
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Figure 6. Induction of GFP under high-light conditions. Expression of GFP was induced under
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low-light (LL, 30 µmol photons m−2 s−1) or high-light (HL, 150 µmol photons m−2 s−1)
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conditions when grown in the presence of nitrate. Western blotting analysis was performed
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using a polyclonal anti-GFP antibody. (A) Cells harboring pCA015 were grown under low-light
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conditions. The cells were then irradiated with low or high light intensities, and GFP was
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induced by aTc for the indicated time. (B) Cells harboring pCA015 or pCA016 were grown
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under low-light conditions. The cells were then irradiated with low or high light intensities, and
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GFP was induced by aTc and/or adenine for the indicated time. * indicates that adenine was
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added 24 h before irradiation with high light intensities.
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PpetE
pCA002
PL03
tetR-LVA
PnirA
gfpmut2
pCA013
PL03
tetR-LVA
PL03
gfpmut2 tip-trxAss
PnirA
pCA004
PL03
PnirA
tetR-LVA
gfpmut2
pCA014
PL03
tetR-LVA
PL03
gfpmut2 TetR aptamer
PnirA
pCA011
tetR-LVA
PL03
PnirA
PL03
gfpmut2
pCA015
PL03
tetR-LVA
PL03
gfpmut2
small asRNA for PnirA-tetR
PpetE
pCA012
tetR-LVA
PL03
TetR aptamer with tRNA scaffold
PnirA
PL03
gfpmut2
pCA016
tetR-LVA
small asRNA for PpetE-tetR
PL03
PL03
gfpmut2 adenine off riboswitch
TetR aptamer with tRNA scaffold
FIgure 1
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40,000 35,000 GFP fluorescence (au)
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30,000 25,000
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aTc 0 20 +N 200 2,000 0 −N (ng/ml)
20,000 15,000 10,000 5,000 0 pCA004 (Control)
#1
pCA011 (small asRNA)
#2
pCA013 (TiP-TrxAss)
#3
pCA014 (TetR aptamer)
pCA015 (TetR aptamer + tRNA scaffold)
#4
#5
Figure 2
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A
pCA004 (Control) aTc
0
2
pCA011 (Small asRNA) 20
200
0
2
20
200
(ng/ml)
anti-TetR
B
pCA004 (Control) Time
0
3
8
24
pCA011 (Small asRNA) 48
72
0
3
8
24
48
72 (h)
anti-TetR
anti-GFP
Figure 3
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A pCA004 (Control) 0
3
8
24
pCA015 (TetR aptamer + tRNA scaffold) 48
72
0
3
8
24
48
72
(h)
anti-GFP
pCA004 (Control)
B
0
3
8
24
pCA015 (TetR aptamer + tRNA scaffold) 48
72
0
3
8
24
48
72
(h)
anti-TetR
anti-GFP
Figure 4
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A
pCA015 (TetR aptamer + tRNA scaffold) Adenine
0
2.5
25
250
pCA016 (pCA015 + adenine off riboswitch) 0
2.5
25
250
(µM)
anti-TetR
B
40,000 35,000 GFP fluorescence (au)
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30,000 25,000
aTc 0 20 +N 200 2,000 0 −N (ng/ml)
20,000 15,000 10,000 5,000 0 Adenine
0
250
(µM)
Figure 5
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A pCA015 (TetR aptamer + tRNA scaffold) LL aTc
HL
20 0
24
48
20 72
0
24
200
48
72
0
24
48
(ng/ml) 72
(h)
anti-GFP
B
pCA015 (TetR aptamer + tRNA scaffold)
pCA016 (pCA015 + adenine off riboswitch)
LL aTc
200
0
200
20
0
0
250
250
250*
Adenine 0
HL
200
24
48
72
0
24
48
72
0
24
48
72
0
24
48
72
0
24
48
200
(ng/ml)
250* 72
0
24
48
(µM) 72
(h)
anti-GFP
#1
#2
#3
#4
#5
Figure 6
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