Designing Synthetic Flexible Gene Regulation Networks Using RNA

Sep 16, 2016 - We used three transcriptional repressor systems that are not part of any natural biological clock to build an oscillating network, term...
0 downloads 0 Views 1004KB Size
Subscriber access provided by CORNELL UNIVERSITY LIBRARY

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Synthetic Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

1

Designing synthetic flexible gene regulation networks using RNA

2

devices in cyanobacteria

3

4

5

Akiyoshi Higoa;b, Atsuko Isua;b, Yuki Fukayaa;b, and Toru Hisaboria;b #

6

a

7

Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503, Japan; bCore Research for

8

Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST),

9

Tokyo 102-0075, Japan

Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of

10

11

#

12

Research, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-Ku, Yokohama

13

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

14

15

16

17

18

1

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

19

ABSTRACT

20

In recent years, studies on the development of gene regulation tools in cyanobacteria have been

21

extensively conducted towards efficient production of valuable chemicals. However, there is

22

considerable scope for improving the economic feasibility of production. To improve a recently

23

reported gene induction system using anhydrotetracycline (aTc)–TetR and an endogenous gene

24

repression system using small antisense RNA in the filamentous nitrogen-fixing

25

cyanobacterium Anabaena sp. PCC 7120 (Anabaena), we constructed a positive feedback loop,

26

in which gfp and a small antisense RNA for tetR are controlled by an aTc-inducible promoter.

27

GFP expression in this improved system was higher and longer than the system lacking tetR

28

repression. In addition, using TetR aptamer and a riboswitch, we succeeded in achieving a

29

superior and longer induction of GFP expression even under high-light conditions. Hence,

30

efficient gene induction systems were established in Anabaena by designing a gene regulation

31

network using RNA-based tools.

32

33

Key words: cyanobacteria, gene regulation system, small RNA, riboswitch

34

2

ACS Paragon Plus Environment

Page 2 of 33

Page 3 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

35

INTRODUCTION

36

By designing artificial genetic circuits, in which regulatory devices are appropriately combined,

37

various types of systems have been created to perform specific tasks in living cells since the

38

first construction of the genetic oscillator1 and toggle switch.2 Inspired by electrical engineering

39

and computer science, researchers working in the field of synthetic biology have produced

40

biological counters,3 Boolean logic gates,4 band-path filters,5 and memory.6 In addition, such

41

synthetic biology approach has been applied to the development of biosensors and production of

42

valuable chemicals7

43

Although cyanobacteria have been expected to play a role in the production of useful

44

substances and biofuels because of their photosynthetic ability,8 lack of genetic devices for

45

efficient gene regulation in these organisms has limited their productivity.9, 10 To overcome this

46

challenge, studies on development of gene regulation tools have been actively conducted in

47

recent years.8 Among them, gene induction system consisting of an inducer, anhydrotetracycline

48

(aTc), and a transcriptional repressor, TetR, showed a wide dynamic range in some

49

cyanobacteria, although photolability of aTc limited the usefulness of this system.11–13 A

50

theophylline riboswitch-based gene induction system has also been successfully used in

51

cyanobacteria.14, 15 In addition, gene repression systems have been developed in cyanobacteria.

52

A small RNA based on an Escherichia coli IS10 RNA-IN/OUT system was used for the

3

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

53

repression of exogenous gfp expression.12 Furthermore, we and the other group succeeded in

54

controlling endogenous gene expression by using artificial small antisense RNA (asRNA)13 and

55

CRISPRi system,16 respectively. However, the number of studies on construction of a genetic

56

network using such gene regulation devices is limited. Recently, Immethun et al. built a

57

two-input AND gate in the unicellular cyanobacterium Synechocystis sp. PCC 6803,17

58

demonstrating the potential of synthetic biology in cyanobacteria.

59

For the industrial production of valuable materials in cyanobacteria, it is important to

60

consider the cost of production. Because synthetic inducers are generally expensive, the amount

61

of these inducers used to induce gene expression should be minimal to reduce production costs.

62

In addition, photolability-related issues of aTc should be solved when TetR system is used in

63

photosynthetic cyanobacteria. Hence, we improved our previous TetR system in Anabaena13

64

using a synthetic biology approach. We designed a positive feedback loop and succeeded in

65

achieving greater and prolonged gene induction using only a small amount of aTc. Furthermore,

66

by combining a TetR aptamer that inactivates TetR repression activity and a riboswitch that

67

terminates transcription in the presence of adenine, we enabled strong and prolonged gene

68

induction under high-light conditions.

69

70

RESULTS AND DISCUSSION

4

ACS Paragon Plus Environment

Page 4 of 33

Page 5 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

71

In our previous study, we demonstrated that the amount of TetR expressed in cells is a

72

determinant of responsiveness to the inducer aTc.13 Thus, tetR was expressed under the control

73

of nirA promoter, which is active in the presence of nitrate and is repressed in the absence of a

74

nitrogen source.18 Therefore, gene expression is induced under nitrogen-fixing conditions13

75

(pCA004 in Figure 1 and supplemental Table S1). In the pCA004 system, induction by nitrogen

76

starvation as well as by aTc was possible. Lowering the TetR amount by the promoter

77

engineering of PnirA facilitated the creation of a strong induction system (pCA00513), which,

78

however, had a narrow dynamic range. In this study, we designed a positive feedback loop to

79

induce gene expression strongly with a wide dynamic range. We combined two systems, TetR

80

induction and small asRNA repression, which were previously developed by us.13 A small

81

asRNA was designed to repress tetR expression, and the small asRNA and the reporter gene gfp

82

were expressed under the control of PL03, which is regulated by aTc and TetR (pCA011 in

83

Figure 1 and supplemental Table S1). In the case of the pCA011 system, once these genes are

84

induced, the amount of TetR in the cell should severely decrease and consequently significant

85

gene induction should occur. Here, we expressed TetR with a protease tag LVA13 at the

86

C-terminus for the efficient turnover of this protein in the cell (Figure 1).

87

We introduced two plasmids, pCA004 that lacked the asRNA gene for tetR repression

88

and pCA011, into Anabaena. We then measured GFP fluorescence in these two strains, which

5

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

89

was induced by indicated concentrations of aTc for 24 h in nitrate-replete (+N) medium (Figure

90

2). In addition, we measured GFP fluorescence induced by nitrogen starvation (−N) for 72 h as

91

well. GFP fluorescence was very low without the inducer in +N conditions in each strain

92

(Figure 2, #1 and #2). As expected, when GFP expression was induced by aTc or nitrogen

93

starvation, the asRNA-expressing strain showed stronger fluorescence than the control strain.

94

However, even in the presence of 2,000 ng/ml aTc, the pCA011-contaning strain showed only

95

one-fourth of GFP fluorescence in a strain without tetR (pCA00113), indicating that a further

96

improvement is possible. Western blotting analysis confirmed that the amount of TetR

97

decreased in response to aTc in the asRNA-expressing strain but not in the control strain (Figure

98

3A). This result demonstrates the plasticity of small asRNA, which can repress a gene in an

99

artificial network as well as an endogenous gene.13 When 20 ng/mL aTc was added, the TetR

100

amount in the asRNA-expressing strain decreased within 24 h. Even after 72 h, the amount of

101

TetR was lower in the asRNA-expressing strain than in the control strain (Figure 3B). In

102

addition, GFP expression continued for 72 h in the former strain, while it gradually decreased

103

after 24 h in the latter strain (Figure 3B). These results indicate that construction of an artificial

104

gene regulatory network complemented photolability of aTc and that such a synthetic biology

105

approach is useful in Anabaena.

106

Next, we wanted to improve the system in which tetR is expressed under the control of

6

ACS Paragon Plus Environment

Page 6 of 33

Page 7 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

107

PpetE, which would be active irrespective of nitrogen source.19 We designed a similar positive

108

feedback loop, in which tetR expression under PpetE is repressed by a small asRNA (pCA012 in

109

Figure 1 and supplemental Table S1). A control plasmid pCA002 and pCA012 were introduced

110

into Anabaena. We confirmed that the amount of TetR decreased in response to aTc in the

111

pCA012-harboring strain (Supplemental Figure S1A). Furthermore, GFP fluorescence in the

112

strain was higher than that in the control strain (Supplemental Figure S1B). These results

113

suggest that the small asRNA approach has a potential to improve a variety of gene expression

114

systems.

115

We then constructed a positive feedback loop, in which the amount of TetR is not

116

affected but the TetR repressor function is inhibited by TetR-inducing peptide (TiP)20 or TetR

117

aptamer.21, 22 A similar design for a prolonged induction had been previously proposed.11 TiP is

118

generally used with scaffold proteins such as TrxA from E. coli.20 In this study, we used E. coli

119

TrxASS, in which two cysteine residues at the active site were substituted with serine to avoid

120

undesirable thiol-disulfide exchange reactions with endogenous proteins in Anabaena.

121

Unexpectedly, compared with the control strain, each Anabaena strain containing

122

Tip-TrxASS-expressing plasmid pCA013 or TetR aptamer-expressing plasmid pCA014 showed

123

no significant differences in GFP fluorescence at various concentrations of aTc (Figure 2, #1, #3,

124

and #4). An LVA tag at the C-terminus of TetR or rapid turnover of TetR may interfere with

7

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

125

stable interaction between TetR and TiP-TrxASS or the TetR aptamer. Next, we expressed the

126

TetR aptamer with a tRNA scaffold23 (pCA015 plasmid in Figure 1 and supplemental Table S1)

127

to stabilize the TetR aptamer in the cells. While GFP fluorescence in the pCA015-containing

128

strain was 5.5-fold higher than that in the control strain under non-induced conditions, the

129

former strain exhibited a 9-fold higher GFP fluorescence when induced under nitrate depletion

130

than that exhibited by the control strain (Figure 2, #1 and #5). In addition, GFP fluorescence in

131

the pCA015-containing strain was higher by induction with, in particular, a small amount of aTc

132

(20 ng/mL) than the fluorescence in the control strain. These results indicate that a strong GFP

133

induction by aTc or nitrate depletion was achieved, while the pCA015 system was slightly leaky

134

compared to the control system.

135

Time-course analysis of GFP induction was performed in a tRNA-scaffold TetR

136

aptamer-expressing strain using pCA015. In this strain, GFP was strongly expressed even after

137

72 h of induction with 20 ng/mL aTc as opposed to the control strain (Figure 4A). When nitrate

138

was depleted from the culture medium in both strains, the amount of TetR clearly decreased

139

after 8 h (Figure 4B). Unexpectedly, GFP gradually accumulated after 24 h of nitrate depletion

140

in these two strains (Figure 4B), in contrast to when GFP was induced by aTc (Figure 4A).

141

Development of rapid induction by decrease of TetR amount is required in the future.

142

However, such a gradual induction may be preferred in some cases because rapid induction of

8

ACS Paragon Plus Environment

Page 8 of 33

Page 9 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

143

some genes and successful modulation of metabolism immediately after nitrate depletion may

144

inhibit the complex heterocyst differentiation process and nitrogen fixation.

145

For constructing a flexible gene induction system, we adopted another approach: a

146

riboswitch was used to control the expression of tetR. Riboswitch xpt(C74U)/metE,24 an off

147

switch that terminates transcription in the presence of adenine, was inserted downstream of PnirA

148

in pCA015. The resultant plasmid pCA016 (Figure 1 and supplemental Table S1) was

149

introduced into Anabaena. In the pCA16-containing strain, addition of adenine decreased the

150

amount of expressed TetR in a concentration-dependent manner (Figure 5A). This result clearly

151

indicates that the riboswitch is functional in Anabaena. The amount of TetR after 24 h

152

incubation with 250 µM adenine was one-tenth of that in the absence of adenine (Figure 5A).

153

Next, GFP was induced by aTc or nitrate depletion both in the presence and absence of adenine.

154

After cells were incubated in the presence or absence of 250 µM adenine for 48 h under +N

155

conditions, aTc was added to the culture medium and GFP expression was induced for 24 h.

156

Furthermore, GFP fluorescence was induced by incubation for 72 h under −N conditions

157

irrespective of the presence of adenine. When adenine was not supplied, GFP expression was

158

induced by aTc in a concentration-dependent manner (Figure 5B). Adenine alone induced GFP

159

fluorescence, and aTc further induced the fluorescence in +N conditions. In addition, adenine

160

alone strongly induced GFP under −N conditions. These results indicate that flexible gene

9

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

161

induction is possible in the pCA016 system by the addition of adequate quantities of two

162

inducers, aTc and adenine, and by the selection of specific growth conditions. Supplemental Figure S2 shows microphotographs of the pCA016 strain. Adenine alone

163

164

induced GFP fluorescence sparsely but strongly across the filaments. GFP fluorescence of the

165

majority of cells was strongly induced by aTc alone or both of aTc and adenine. These results

166

suggest that the pCA016 induction system exhibits all-or-none response rather than a graded

167

one.

168

When aTc is used as an inducer in gene induction systems, photolability of this molecule

169

may be problematic, especially under high-light conditions. Indeed, 20 ng/mL aTc hardly

170

induced GFP expression in the pCA015-harboring strain under high-light (150 µmol photons

171

m−2 s−1) conditions as opposed to the results under low-light (30 µmol photons m−2 s−1)

172

conditions (Figure 6A). Addition of 200 ng/mL aTc induced GFP expression for a short period

173

(24 h) in the strain under high-light conditions but at a lesser extent than that induced under

174

low-light conditions with addition of 20 ng/mL aTc. To overcome these photolability-related

175

problems observed in aTc, we examined the adjusted induction conditions by decreasing the

176

amount of TetR via addition of adenine in the pCA016-containing strain. Addition of only 200

177

ng/mL aTc hardly induced GFP expression under high-light conditions (Figure 6B, #1). In

178

contrast, addition of 250 µM adenine slightly induced GFP after 72 h under high-light

10

ACS Paragon Plus Environment

Page 10 of 33

Page 11 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

179

conditions in this strain (Figure 6B, #2). When both aTc and adenine were added, GFP

180

expression was induced after 24 h and its expression continued until 72 h (Figure 6B, #3). When

181

adenine was added 24 h before the addition of aTc and irradiation with high light intensities, 20

182

or 200 ng/mL aTc induced GFP expression to a greater extent (Figure 6B, #4, and #5) than that

183

induced when aTc and adenine were added simultaneously.

184

The results shown in Figure 6B indicate that construction of an artificial gene regulatory

185

network enabled gene induction with a wide dynamic range in the aTc-TetR system even under

186

high-light conditions. The theophylline riboswitch system14, 15 may be alternatively used for

187

gene induction under high-light conditions because theophylline is insensitive to light. However,

188

the theophylline riboswitch system shows a narrow dynamic range compared with the aTc-TetR

189

system (Supplemental Figure S3). In addition, a functional RNA gene could not be induced by

190

the theophylline riboswitch system, in which translation is activated in the presence of

191

theophylline.14, 15

192

In the present study, we demonstrated that purpose-dependent gene regulatory networks

193

could be created by simple combination of gene regulation components, TetR and RNA-based

194

devices. Complex gene regulation networks consisting of non-coding RNAs and proteins occur

195

ubiquitously in nature. For example, similar to the TetR aptamer that sequesters TetR,22 a small

196

non-coding RNA, whose function is controlled by a riboswitch, sequesters a regulator protein to

11

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

197

prevent expression of ethanolamine utilization genes in Firmicutes. 25, 26 Another example was

198

recently demonstrated in the unicellular cyanobacterium Synechocystis sp. PCC 6803: a

199

feed-forward loop consisting of a transcriptional regulator and a small non-coding RNA that

200

prevents translation of target mRNAs is essential for rapid acclimation response to high-light

201

stress.27 In addition, development of RNA-based gene regulation tools has been actively pursued

202

in cyanobacteria over the past few years12–16, 28, 29 because of their ease of design.30 Inspiration

203

from natural systems, as well as electrical engineering and computer science, should make it

204

possible to create various types of gene regulation systems, in which genes of interest are

205

robustly controlled even under outdoor conditions, by using especially RNA-based devices.

206

Construction of such gene regulation networks would help to generate valuable products using

207

cyanobacteria in the future.

208

209

METHODS

210

Bacterial strains and growth conditions

211

Anabaena strains were routinely grown at 30°C at 30–35 µmol photons m−2 s−1 in BG11

212

medium31 supplemented with 20 mM HEPES-NaOH (pH 7.5) and 5 µg/mL neomycin sulfate

213

unless otherwise stated. For nitrogen starvation experiments, cells were grown in the same

214

medium lacking NaNO3 (BG110). Liquid culture was bubbled with air containing 1.0% (v/v)

12

ACS Paragon Plus Environment

Page 12 of 33

Page 13 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

215

CO2.

216

217

Plasmid construction

218

DNA fragments were inserted between the EcoRI and BamHI sites of pRL25c,32 a shuttle vector

219

replicating in Anabaena (Figure 1). Detailed sequences are described in the Supplemental

220

Information.

221

222

Measurement of GFP fluorescence

223

GFP fluorescence from Anabaena cultures was measured as described previously.13 Aliquots (3

224

ml) of log-phase cultures (OD750 = 0.1–0.2, 48 h after inoculation) of GFP-expressing Anabaena

225

strains were moved into 12-well plates with the indicated amount of aTc or with theophylline

226

and 10 mM bicarbonate and then incubated for 24 h at 30–35 µmol photons m−2 s−1. Then,

227

fluorescence intensities of GFP expressed in Anabaena cultures (OD750 = 0.2–0.4) were

228

measured using a fluorescence spectrophotometer FP-8500 (JASCO, Tokyo, Japan) after the

229

optical density of Anabaena at 750 nm (OD750) was adjusted to 0.1–0.12 with BG11 or BG110

230

medium supplemented with 20 mM HEPES-NaOH (pH 7.5). An excitation wavelength of 488

231

nm and an emission wavelength of 510 nm were used. To obtain the expression level in the

232

GFP-expressing Anabaena strains, the fluorescence intensity of the cells containing the empty

13

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

233

vector was subtracted as the background.

234

235

Total protein extraction

236

Cultures of Anabaena strains were centrifuged at 4°C at 2,000 g for 3 min. The cell pellets were

237

frozen in liquid nitrogen and stored at −80°C until use. The cells were re-suspended in SDS

238

sample buffer [125 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 6% (v/v)

239

2-mercaptoethanol, and 0.01% (w/v) bromophenol blue], and immediately boiled for 5 min.

240

After centrifugation at 20,000 g for 10 min, the supernatants were collected as total protein.

241

Protein concentration was determined with a CB-X protein assay kit (G-Biosciences, St. Louis,

242

MO, USA) using BSA as a standard.

243

244

Western blotting analysis

245

Equal amounts of total protein (5 µg and 200 ng for detection of TetR and GFP, respectively)

246

obtained from various cell lysates were separated on a denaturing SDS-PAGE gel and blotted

247

onto the PVDF membrane. TetR and GFP were detected using a TetR monoclonal antibody

248

(Takara Bio, Kusatsu, Japan) and a GFP polyclonal antibody (Sigma-Aldrich, St. Louis, MO,

249

USA), respectively, with a chemiluminescent kit (ImmunoStar LD, Wako, Osaka, Japan).

250

14

ACS Paragon Plus Environment

Page 14 of 33

Page 15 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

251

Fluorescence microscopy

252

Fluorescence images were taken on a fluorescence microscopy (model IX73, Olympus, Tokyo,

253

Japan) with a mirror unit U-FBNA and U-FGW, for observation of GFP fluorescence and

254

phycobiliprotein autofluorescence, respectively. Cultures were bubbled with air containing

255

1.0% (v/v) CO2.

256

257

258

Supporting Information

259

A document describing the DNA sequences used in this study, as well as summary of aTc-TetR

260

induction systems used in this study in Table S1 and Figure S1-S3.

261

262

Abbreviations: asRNA, antisense RNA; aTc, anhydrotetracycline; GFP, green fluorescent

263

protein; TiP, TetR-inducing peptide.

264

265

Acknowledgments

266

We thank the Biomaterial Analysis Center at the Tokyo Institute of Technology for helping us

267

with DNA sequencing technique. This work was supported by the Core Research of Evolutional

268

Science and Technology program (CREST) at the Japan Science and Technology Agency (JST).

15

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

269

270

Author Contributions

271

AH performed the experiments and data analysis. AI, YF, and TH assisted with the project. AH

272

prepared the initial draft of the manuscript, and AH and TH edited the manuscript.

273

274

Notes

275

The authors declare no competing financial interest.

276

277

16

ACS Paragon Plus Environment

Page 16 of 33

Page 17 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

278

REFERENCES

279

280

281

(1) Elowitz, M. B. and Leibler, S. (2000) A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338.

282

283

284

(2) Gardner, T. S., Cantor, C. R., and Collins, J. J. (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342.

285

286

287

(3) Friedland, A. E., Lu, T. K., Wang, X., Shi, D., Church, G., and Collins, J. J. (2009) Synthetic gene networks that count. Science 324, 1199–1202.

288

289

290

(4) Guet, C. C., Elowitz, M. B., Hsing, W., and Leibler, S. (2002) Combinatorial synthesis of genetic networks. Science 296, 1466–1470.

291

292

(5) Muranaka, N. and Yokobayashi, Y. (2010) A synthetic riboswitch with chemical band-pass

293

response. Chem Commun (Camb) 46, 6825–6827.

294

295

(6) Fritz, G., Buchler, N.E., Hwa, T., and Gerland, U. (2007) Designing sequential transcription

17

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

296

logic: a simple genetic circuit for conditional memory. Syst Synth Biol 1, 89–98.

297

298

(7) Khalil, A. S. and Collins, J. J. (2010) Synthetic biology: applications come of age. Nat Rev

299

Genet 11, 367–379.

300

301

(8) Case, A. E. and Atsumi, S. (2016) Cyanobacterial chemical production. J Biotechnol 231,

302

106–114.

303

304

(9) Berla, B. M., Saha, R., Immethun, C. M., Maranas, C. D., Moon, T. S., and Pakrasi, H. B.

305

(2013) Synthetic biology of cyanobacteria: unique challenges and opportunities. Front.

306

Microbiol. 4, 246.

307

308

(10) Nozzi, N. E., Oliver, J.W. K., and Atsumi, S. (2013) Cyanobacteria as a platform for

309

biofuel production. Front. Bioeng. Biotechnol. 1, 7.

310

311

(11) Huang, H.H. and Lindblad, P. (2013) Wide-dynamic-range promoters engineered for

312

cyanobacteria. J. Biol. Eng. 7, 10.1186/1754–1611–7–10.

313

18

ACS Paragon Plus Environment

Page 18 of 33

Page 19 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

314

(12) Zess, E. K., Begemann, M. B., and Pfleger, B. F. (2015) Construction of new synthetic

315

biology tools for the control of gene expression in the cyanobacterium Synechococcus sp. strain

316

PCC 7002. Biotechnol Bioeng. 113, 424–432.

317

318

(13) Higo, A., Isu, A., Fukaya, Y., and Hisabori, T. (2016) Efficient gene induction and

319

endogenous gene repression systems for the filamentous cyanobacterium Anabaena sp. PCC

320

7120. Plant Cell Physiol. 57, 387–396.

321

322

(14) Nakahira, Y., Ogawa, A., Asano, H., Oyama, T., and Tozawa, Y. (2013)

323

Theophylline-dependent riboswitch as a novel genetic tool for strict regulation of protein

324

expression in cyanobacterium Synechococcus elongatus PCC 7942. Plant Cell Physiol. 54,

325

1724–1735.

326

327

(15) Ma, A. T., Schmidt, C. M., and Golden, J.W. (2014) Regulation of gene expression in

328

diverse cyanobacterial species by using theophylline-responsive riboswitches. Appl. Environ.

329

Microbiol. 80, 6704–6713.

330

331

(16) Yao, L., Cengic, I., Anfelt, J, and Hudson, E. P. (2016) Multiple gene repression in

19

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

332

cyanobacteria using CRISPRi. ACS Synth Biol 5, 207–212.

333

334

(17) Immethun, C. M., Ng, K. M., DeLorenzo, D. M., Waldron-Feinstein, B., Lee, Y. C., and

335

Moon, T. S. (2016) Oxygen-responsive genetic circuits constructed in Synechocystis sp. PCC

336

6803. Biotechnol Bioeng. 113, 433–442.

337

338

(18) Frías, J. E. and Flores, E. (2010) Negative regulation of expression of the nitrate

339

assimilation nirA operon in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC

340

7120. J. Bacteriol. 192, 2769–2778.

341

342

(19) Buikema, W. J. and Haselkorn, R. (2001) Expression of the Anabaena hetR gene from a

343

copper-regulated promoter leads to heterocyst differentiation under repressing conditions. Proc.

344

Natl. Acad. Sci. USA 98, 2729–2734.

345

346

(20) Klotzsche, M., Berens, C., and Hillen, W. (2005) A peptide triggers allostery in Tet

347

repressor by binding to a unique site. J. Biol. Chem. 280, 24591–24599.

348

349

(21) Hunsicker, A., Steber, M., Mayer, G., Meitert, J., Klotzsche, M., Blind, M., Hillen, W.,

20

ACS Paragon Plus Environment

Page 20 of 33

Page 21 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

350

Berens, C., and B., Suess. (2009) An RNA aptamer that induces transcription. Chem Biol. 16,

351

173–180.

352

353

(22) Steber, M., Arora, M., Hofmann, J., Brutschy, B., and Suess, B. (2011) Mechanistic basis

354

for RNA aptamer-based induction of TetR. Chembiochem. 12, 2606-2614

355

356

(23) Ponchon, L and Dardel, F. (2007) Recombinant RNA technology: the tRNA scaffold. Nat

357

Methods 4, 571–576.

358

359

(24) Ceres, P., Garst, A. D., Marcano-Velázquez, J.G., and Batey, R. T. (2013) Modularity of

360

select riboswitch expression platforms enables facile engineering of novel genetic regulatory

361

devices. ACS Synth Biol 2, 463–472.

362

363

(25) DebRoy, S., Gebbie, M., Ramesh, A., Goodson, J. R., Cruz, M. R., van Hoof, A., Winkler,

364

W. C., and Garsin, D. A. (2014) Riboswitches. A riboswitch-containing sRNA controls gene

365

expression by sequestration of a response regulator. Science 345, 937-940.

366

367

(26) Mellin, J. R., Koutero, M., Dar, D., Nahori, M. A., Sorek, R., and Cossart, P. (2014)

21

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

368

Riboswitches. Sequestration of a two-component response regulator by a riboswitch-regulated

369

noncoding RNA. Science 345, 940-943.

370

371

(27) Kadowaki, T., Nagayama, R., Georg, J., Nishiyama, Y., Wilde, A., Hess, W. R., Hihara, Y.

372

(2016) A feed-forward loop consisting of the response regulator RpaB and the small RNA

373

PsrR1 controls light acclimation of photosystem I gene expression in the cyanobacterium

374

Synechocystis sp. PCC 6803. Plant Cell Physiol. 57, 813-823

375

376

(28) Abe, K., Sakai, Y., Nakashima, S., Araki, M., Yoshida, W., Sode, K., and Ikebukuro, K.

377

(2014) Design of riboregulators for control of cyanobacterial (Synechocystis) protein expression.

378

Biotechnol. Lett. 36, 287–294.

379

380

(29) Sakai, Y., Abe, K., Nakashima, S., Ellinger, J.J., Ferri, S., Sode, K., and Ikebukuro, K.

381

(2015) Scaffold-fused riboregulators for enhanced gene activation in Synechocystis sp. PCC

382

6803. MicrobiologyOpen 4, 533–540.

383

384

(30) Peters, G., Coussement, P., Maertens, J., Lammertyn, J., and De Mey, M. (2015) Putting

385

RNA to work: Translating RNA fundamentals into biotechnological engineering practice.

22

ACS Paragon Plus Environment

Page 22 of 33

Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

386

Biotechnol Adv 33, 1829–1844.

387

388

(31) Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., and Stanier, R. Y. (1979) Generic

389

assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol.

390

111, 1–61.

391

392

(32) Wolk, C. P., Cai, Y., Cardemil, L., Flores, E., Hohn, B., Murry, M., Schmetterer, G.,

393

Schrautemeier, B., and Wilson, R. (1988) Isolation and complementation of mutants of

394

Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen. J. Bacteriol. 170,

395

1239–1244.

396

23

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

397

Figure legends

398

Figure 1. The basic design of plasmids used in this study.

399

400

Figure 2. Induction of gfp expression by aTc or nitrate depletion through synthetic gene

401

regulation networks. Anabaena cells were treated with different concentrations of aTc for 24 h

402

under nitrate-replete conditions (+N) or were grown under nitrogen starvation conditions (−N)

403

for 72 h; GFP fluorescence was then measured and normalized (optical density at 750 nm). The

404

experiments using each plasmid were labeled #1 to #5 in the figure. Data represent the mean ±

405

SD (n = 3 from independent cultures).

406

407

Figure 3. Repression of TetR by small asRNA. (A) Dependence of TetR amount on aTc

408

concentrations. Different concentrations of aTc were added to the medium. After 24 h, proteins

409

from each strain grown under +N conditions were extracted, and TetR was detected using a

410

monoclonal anti-TetR antibody by western blotting. (B) Time-course analysis of TetR and GFP

411

amount after the addition of 20 ng/mL aTc. Total proteins from each strain grown under +N

412

conditions were extracted after the indicated time and analyzed by western blotting.

413

414

Figure 4. Induction of GFP in a TetR aptamer-expressing strain. (A) Time-course analysis of

24

ACS Paragon Plus Environment

Page 24 of 33

Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

415

GFP amount after the addition of 20 ng/mL aTc when the strain was grown under +N conditions.

416

Total proteins from each strain were extracted after the indicated time and analyzed by western

417

blotting. (B) Time-course analysis of TetR and GFP amount after nitrogen starvation. Total

418

proteins from each strain were extracted after the indicated time and analyzed by western

419

blotting.

420

421

Figure 5. Induction of GFP by aTc and adenine with an adenine off riboswitch. (A) The amount

422

of TetR in strains harboring pCA015 or pCA016 with different concentrations of adenine under

423

+N conditions. Western blotting analysis was performed using a monoclonal anti-TetR antibody.

424

(B) Anabaena strain harboring pCA016 was treated with different concentrations of aTc for 24

425

h under nitrate-replete conditions (+N) or was grown under nitrogen starvation conditions (−N)

426

for 72 h in the absence or presence of 250 µM adenine. GFP fluorescence was then measured

427

and normalized (optical density at 750 nm). Data represent the mean ± SD (n = 3 from

428

independent cultures).

429

430

Figure 6. Induction of GFP under high-light conditions. Expression of GFP was induced under

431

low-light (LL, 30 µmol photons m−2 s−1) or high-light (HL, 150 µmol photons m−2 s−1)

432

conditions when grown in the presence of nitrate. Western blotting analysis was performed

25

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

433

using a polyclonal anti-GFP antibody. (A) Cells harboring pCA015 were grown under low-light

434

conditions. The cells were then irradiated with low or high light intensities, and GFP was

435

induced by aTc for the indicated time. (B) Cells harboring pCA015 or pCA016 were grown

436

under low-light conditions. The cells were then irradiated with low or high light intensities, and

437

GFP was induced by aTc and/or adenine for the indicated time. * indicates that adenine was

438

added 24 h before irradiation with high light intensities.

439

26

ACS Paragon Plus Environment

Page 26 of 33

Page 27 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

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

ACS Paragon Plus Environment

ACS Synthetic Biology

40,000 35,000 GFP fluorescence (au)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

30,000 25,000

Page 28 of 33

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

ACS Paragon Plus Environment

Page 29 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

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

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

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

ACS Paragon Plus Environment

Page 31 of 33

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)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

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

ACS Paragon Plus Environment

ACS Synthetic Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 33

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

ACS Paragon Plus Environment

Page 33 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Synthetic Biology

Graphic Table of Contents 35x23mm (600 x 600 DPI)

ACS Paragon Plus Environment