From small cyanobacterial RNA regulators to synthetic RNA device

RNA Design Workshop 2015 From small cyanobacterial RNA regulators to synthetic RNA devices 09-07-2015

Institute of Synthetic Microbiology

Dennis

Ilka Sabine

Regulatory RNA

Anika

Metabolic Engineering

Cyanobacteria

Circadian Clock

Cyanobacterial diversity

I

II David M. Kehoe PNAS 2010;107:9029-9030

http://kids.britannica.com/compton s/art-190947/Cyanobacteria-suchas-the-organisms-living-in-this-hotspring

Dagan et al. 2013

http://celebrat ing200years.n oaa.gov/found ations/coastal _research/pot omac_river_6 50.html Flombaum et al. 2013

http://www.polar trec.com/expediti ons/prehistorichuman-responseto-climatechange/journals/ 2009-03-23

IV

V

http://www.science20.com/causes_effects_and_manage ment_of_blue_green_algae_cyanobacteria_and_their_ha rmful_algal_blooms_in_australian_water-139030

www.ibvf.cartuja.csic.es/ Cultivos/Seccion.htm (2009)

III

Cyanobacterial biotechnology

Ethanol production at Algenol (Florida)

http://www.algenol.com/direct-to-ethanol/direct-to-ethanol

Front Bioeng Biotechnol. 2014; 2: 22.

Small RNA regulators (sRNA) in Synechocystis 314 ig-ncRNAs 1011 asRNAs for 866 genes  25% of all genes contain asRNAs

log2 read count

732 iRNAs

Natural RNA regulators in cyanobacteria Example: trans-encoded sRNA PsrR1 PsrR1 accumulates under High Light & Ci limitation

PsrR1

psaL PsrR1

Northern Blot

Anti-SD

Mitschke, Georg, Scholz, Sharma, Dienst, Bantscheff, Voß, Steglich, Wilde, Vogel, Hess. PNAS 2011 Georg, Dienst, Schürgers, Kuchmina, Wallner, Klähn, Knoop, Lokstein, Hess, Wilde. Plant Cell 2014

Natural RNA regulators in cyanobacteria Example 1: trans-encoded sRNA PsrR1 PsrR1 targets psaL mRNA

EMSA Mitschke, Georg, Scholz, Sharma, Dienst, Bantscheff, Voß, Steglich, Wilde, Vogel, Hess. PNAS 2011 Georg, Dienst, Schürgers, Kuchmina, Wallner, Klähn, Knoop, Lokstein, Hess, Wilde. Plant Cell 2014

Small RNA regulators (sRNA) in Synechocystis SyR1/ PsrR1 (I)

Mutant characterization (Physiology)

(II)

Biocomputational Target Prediction

(III)

Experimemntal target detection – microarray upon pulsed OE -

(IV)

Validation Experiments

(V)

Mechanistical characterization Georg, Dienst, Schürgers, Kuchmina, Wallner, Klähn, Knoop, Lokstein, Hess, Wilde. Plant Cell 2014

Cyanobacterial biotechnology Our Goal – Controlled Synthesis of triterpenes in Synechocystis PCC 6803 Working platform

Goal 1 – Stable Synthesis of marneral, thalianol and/or β-amyrin…

… and further triterpene candidates

Cyanobacterial biotechnology Our Goal – Controlled Synthesis of triterpenes in Synechocystis PCC 6803

Strategy - extended comparator → self-adjustment & balancing oxidosqualene and marneral biosynthesis

The Ribonets Project Synthetic RNA signaling networks

www.ribonets.eu www.ribonets.eu

Reporter system for comparator device

YFP

yfp

Input signals

CFP cfp

RNAdev 2 E. coli

YFP

RNAdev 1

IPTG

yfp

+

+

cfp

cfp E. coli

CFP

Output signals

+ +

ATc RNAdev comparator

yfp

Candidate RNAdev for in vivo analysis In total 6 RNAdev candidates:

Direct OFF

Switch C99 and F34

Direct ON

Switch A94 and E63

Indirect OFF

Switch D50 and H60

Modular Cloning of RNA devices

Northern analysis of transRNA expression

Northern analysis of transRNA expression

53 nt  130 nt

End-point mVenus measurements IPTG/ aTc as input signals

Indirect OFF switch: D50



always ON

Indirect OFF switch: H60



always OFF

End-point mVenus measurements IPTG/ aTc as input signals

Direct OFF switch: C99 

always ‚ON‘

RBS

• ~3-4 –fold reduced mVenus accumulation (cf. D50, F34, E63) cisC99

cisD50

End-point mVenus measurements IPTG/ aTc as input signals



0 aTc

40000 35000

F34

30000

EVC

25000

F34-T7

always ON

20000 15000 10000

35000

F34

30000

EVC

25000

F34-T7

20000 15000 10000

5000

5000

0

0

0 µM IPTG

100 aTc

40000

Venus fluorescence/ OD600

Venus fluorescence/ OD600

Direct OFF switch: F34/ F34-T7

200 µM IPTG

0 µM IPTG

200 µM IPTG

End-point mVenus measurements IPTG/ aTc as input signals

Venus fluorescence/ OD600

Direct ON switch: F34  160000

always ON

E63_0 IPTG

EVC_0 IPTG

E63_200 IPTG

EVC_200 IPTG

E63-T7

140000 120000 100000

80000 60000 40000 20000 0

0 aTc

100 aTc

200 aTc

Northern validation of transRNA/mRNA expression … preliminary data transF34

0 0 100 200

200 0 100 200

500 0 100 200

µM IPTG ng

mL-1

aTc

transC99

0 0

transF34-T7 0

200 100

0

100

0

200 100

0

100

µM IPTG Cng mL-1 aTc  131 nt  101 nt

mVenus

mVenus

Candidate RNAdev tested in vivo cisRNA / transRNA

F34

Always ON

C99

‚Always ON‘

Switch

E63

Always ON

Indirect OFF Switch

D50

Always ON

Direct OFF Switch

Direct ON

H60

???

‚Always OFF‘

Structural probing: SHAPE-seq



Kit-like protocol

Structural probing: SHAPE-seq Establishing SHAPE-seq 2.0 in Düsseldorf/ Jülich • Four transRNA candidates due to design • Four transRNA candidates + T7-Terminator from pRSF plasmid • Four cisRNA candidates + 18 nt of mVenus reporter gene • Aptamer 14 and 210 (RNA- Selex/ Sabine)

• Several native sRNA from E. coli, Synechocystis 6803, C. glutamicum

Tino Polen

Structural probing: SHAPE-seq … preliminary data #10 – cisD50 +18 Full-lenght: 226 bp

#12 – cisH60 +18 Full-lenght: 226 bp ELECTROPHEROGRAMS 12 PCR CYCLES

ELECTROPHEROGRAMS 12 PCR CYCLES

1550

C10: #10 - NMIA -12cycles

C12: #12 - NMIA -12cycles

1350

1450

D10: #10 - DMSO -12cycles

D12: #12 - DMSO -12cycles

RFU

1150

RFU

Full-lenght

1950

1750

950 750

950

Full-lenght

550

450

350 150 -50 100

120

140

160

180

200

SIZE (BP)

220

240

260

280

300

-50 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

SIZE (BP)

Structural probing: SHAPE-seq Aptamer210

Electropherograms Apt210 +ligand 12 PCR cycles

Full-lenght: 223 bp 4990

Full-lenght

RFU

3990

2990

A1: #Apt210- NMIA_12 cycles 1990

A2: #Apt210- DMSO_12 cycles 990 -10 100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

280

290

300

Size (bp)

Electropherograms Apt210 -ligand 12 PCR cycles

1990

Full-lenght

RFU

1490

A3: #Apt210+ NMIA_12 cycles

990

A4: #Apt210+ DMSO_12 cycles 490

-10 100

110

120

130

140

150

160

170

180

190

200

210

Size (bp)

220

230

240

250

260

270

280

290

300

Establishing a cyanobacterial reporter system Example: comparator device in vivo analysis … in Cyanobacteria

yfp

RNAdev comparator

Input signals

ATc

RNAdev 2

RNAdev 1

IPTG

+ +

cfp

yfp zFP cfp

Synechocystis

Output signals

+ +

xFP

?

xFP

yfp

cfp Synechocystis

zFP

?

Establishing a cyanobacterial reporter system Example: comparator device in vivo analysis … in Cyanobacteria

yfp

RNAdev comparator

Input signals

ATc

RNAdev 2

RNAdev 1

IPTG

+ +

cfp

yfp zFP cfp

Synechocystis

Output signals

+ +

xFP

?

xFP

yfp

cfp Synechocystis

zFP

?

Establishing a cyanobacterial reporter system Cu2+

+

PpetJ

PrnpB

trRAJ11

mVenus Fluorescence (Ex.585nm/Em.540nm * OD750 -1)

25000

cisRAJ11

mVenus

[Cu2+] ↑

20000 15000 10000 5000 0

pRAJ11 -Cu

pVZ-spec -Cu

pRAJ11 1xCu

pVZ-spec 1xCu

pRAJ11 2.5xCu

pVZ-spec 2.5xCu

pRAJ11 5xCu

pVZ-spec 5xCu

Acknowledgements Ilka M. Axmann Sabine Schneider Jan-Philipp Kunz (JPK) Michelle Heinen Oliver Klaus Janos Jablonski Nic Schmelling Vanessa Hueren … and collaborators: TBI Vienna: Sven Findeiß Stefan Hammer Christoph Flamm

CNRS Paris: André Estévez-Torres Jonathan Lee Tin Wah

15/ 20

FZ Jülich: Tino Polen Ulli Degner

IMET Jülich: Thomas Drepper Anita Loeschcke

Uni Freiburg: Jens Georg Annegret Wilde Wolfgang R. Hess

Thank You for your attention

trans-D50 + terminator

trans-D50 + terminator

trans-C99 - terminator

trans-D50 - terminator

trans-F34 + terminator

trans-H60 + terminator

trans-F34 - terminator

trans-H60 - terminator