Our Team The RNA engineering issue

RNA parts are: Powerful Programmable However： No standard protocol

Kim, J. N.; Breaker, R. R., Biology of the Cell 2008, 100, (1), 1-11. Our goal

A Standard Protocol For Riboswitches • For convenience • For better RNA synthetic biology Five step process The first instructions

Tip: unpublished works are highlighted

Input

Set X = Aleader Discovered in AAC and AAD The most complex riboswitch

Input The most complex riboswitch • Bi-cistron • ORF-11 • attI sites

Input The most complex riboswitch • Bi-cistron • ORF-11 • attI sites • Aptamer

Jia Xu et al. Cell 2013 Definition

Spinach-Test • To visualize the in vivo conformational switches • RNA-mimic GFP: Spinach • The necessary and replaceable Stem loop 2 • Fusing the Spinach and X

J S Paige et al. Science 2012 Estimation Quantitative estimation based on promoter library

14 J23100 J23101 J23105 J23110 Promoter 5’UTR Reporter 12 10

8 6 RFU RFU (A.U.) 4 2 0

-7 -6 -5 -4 -3

log10 Kana (umol/L) Estimation Response Curve Analysis • Dose-response curve • Performance descriptor – Basal level – Highest level – Dynamic range – Working range

Confocal image Modification & Optimization

The traditional method: Mutagenesis based on the design principle of riboswitch

k f K K + K (1+ K L) P = A 1 B 2 ap a1K1 + a2 (1+ K2L) P: protein production kf: transcriptional initiation rate

KA,KB: representative regulatory activities of conformation A and B

a1,a2,ap: degradation rate of conformation A and B, and protein K1: partitioning constant K2: the aptamer association constant L: ligand concentration Modification & Optimization

Modeling of the riboswitches • Increasing the relative stability of conformation A

• Increasing the binding affinity

Modification & Optimization

Mut-3 Mut-8 1.42 Practice: 1.34 1.40 1.32 1.38 Mutants of Aleader 1.30 RFU RFU (A.U.) RFU (A.U.) 1.36 • We mutated the ALeader 1.28 1.34 1.26 1.32

• And estimate the mutants 1.24

-6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5

log10 [Kana] (umol/L) log10 [Kana] (umol/L) Mut-9 Mut-10 Mut-11 1.38 1.40 1.40 1.36 1.38 1.38 1.36 1.36 1.34 1.34 RFU RFU (A.U.) RFU RFU (A.U.) RFU (A.U.) 1.34 1.32 1.32 1.32 1.30 1.30 1.28 1.30

-6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5

log10 [Kana] (umol/L) log10 [Kana] (umol/L) log10 [Kana] (umol/L) Modification & Optimization If mutants fits our need? • For such a conserve riboswitch • Unworkable

Modification & Optimization n Our strategy: K K + K (1+ K L) k ∑ Ai 1i B 2 Multi-phase riboswitch P = f i=1 a n p ∑a1iK1i + a2 (1+ K2L) i=1 P: protein production kf: transcriptional initiation rate

KAi,KB: representative regulatory activities of conformation Ai and B

a1i,a2,ap: degradation rate of conformation Ai and B, and protein K1i: partitioning constant between conformation Ai and B

K2: the aptamer association constant L: ligand concentration Modification & Optimization

25 Why it is good? triphase dynamic range biphase dynamic range

Upperlimit of triphase • The First! 20 riboswitch’s dynamic range

Triphase riboswitch can • The Best! break the upperlimit of 15 biphase riboswitch – Always better Upperlimit of biphase – Breaking through the upper limit riboswitch’s dynamic range 10 – Integrating the multi-functions Dynamic range ratio 5

0 −4 −2 0 2 4 10 10 10 10 10 K2

Modification & Optimization Practice: ALeaderT Make an artificial conformation by adding a competitive sequence !

Exposed SD2 Functional terminator Modification & Optimization

Practice: ALeaderT Design • Three states: • Ligand-free – A: Translation repression – B: Termination • Ligand-binding – C: Translation initiation ! Modify and Optimize

12 J23100 J23101 J23104 J23110

10 R1051 With R0010 a promoter library 8

and mRFP1 6 4 Expression level (A.U.) level Expression 2 0

-6.0 -5.5 -5.0 -4.5 -4.0

log10 Kana (umol/L) Modify and Optimize

If ALeaderT fits our need? – lower basal level and higher dynamic range – lower noise Modify and Optimize

pSB1C3-J23100-loci-X-mRFP1

• ALeaderT integrates the Aleader

12 AleaderT terminator and translational AleaderT-BCD

element 10 8 6 Expression level Expression 4 2

-6.0 -5.5 -5.0 -4.5 -4.0

log10 Kana (umol/L) Modify and Optimize

Our strategy：Triphase riboswitch Summary – the advantages of riboswitch – Easily designed – Decreasing the basal level – Improving the dynamic range – Reducing the noise – Integrating the functions Modify and Optimize

• Environment Transformer – Manage the working range rather than extend it Modify and Optimize

• Auto-regulated Transformer Modify and Optimize

• Summary: – Adding competitive sequence è Triphase riboswitch design – Expressing an enzyme è Environ-Transformer – Site-directed mutagenesis Apply

• What about the Kanamycin detector? – High dynamic range – Wide working range

Kanamycin detector = ALeaderT + KanaR User guides for Application To Users: B0015 Terminator Efficiency 1.0 Be careful of RNA interaction Interacted Normal

Interfered terminator 0.8 0.6 Test Input 0.4 Terminator Efficiency Terminator 0.2 Test Output 0.0 Interacted CC JK Future works

The different behaviors of RNA and protein regulators in the networks Human Practice

• iGEMcyclopedia 2.0 – For fresh iGEMers – For better communication Human Practice

Activities at Fudan and Communications with other teams

With association of Bertalanffy With iGEM2013_USTC CHINA Human Practice

• Spread the idea of iGEM – The communication with Prof Chen and Prof Murchie’s Lab Judge Form

• Best Human Practice: • Best new Biobrick part, Natural: iGEMpedia 2.0 BBa_K1100000 (ALeader) • Best Biobrick Measurement • Best new Biobrick part, Engineered: approach: interacted terminator measurement BBa_K1100002 (ALeaderT-BCD), BBa_K1100014(insulator-ALeaderT), • Best New standard: the standard protocol of riboswitch BBa_K1100011(interacted B0015 engineering measurement) • Best Model: • Most improved registry part: the theoretical study of triphase BBa_B0015 (measured by riboswitch BBa_K1100011) • Best Part collection: BBa_K1100140-150 (ALeaderT library) Sponsor and Acknowledgement

• School of life science, Fudan University • Prof. Zhao Guoping, School of Life Science, Fudan University • Prof. Dongrong Chen and Prof. Alastair I.H. Murchie’s Lab， IBS Fudan University • Prof. Lu Daru, School of Life Science, Fudan University • Prof. Zhong Jiang, School of Life Science, Fudan University • Prof. Yang Ji, School of Life Science, Fudan University • Prof. Lv Hong, School of Life Science, Fudan University Associate Prof. • Yu Yao, School of Life Science, Fudan University Associate • Prof. Ding Yu, School of Life Science, Fudan University • Dr. Qi Lei, Center for system and Synthetic Biology, UCSF

Supplementary

Our strategy: Triphase riboswitch

k f K K + K K + K (1+ K L) P = A 2 B 3 C 4 ap KAK2 + KBK3 + KC (1+ K4L) P: protein production kf: transcriptional initiation rate

KA,KB,Kc: representative regulatory activities of conformation A, B and C

a1, a2, a3, ap: degradation rate of conformation A, B, C and protein K1, K2, K3: partitioning constant between conformation A and B, A and C, B and C

K4: the aptamer association constant L: ligand concentration Supplementary Supplementary Supplementary

100

90 triphase riboswitch biphase riboswitch 80 a2/KB = 2

70 e g

n 60 a R

c 50 i m

a 40 n y

D 30

20

10

0 0 1 2 3 4 5 6 7 8 9 10 a1/KA Supplementary Supplementary Supplementary Supplementary Supplementary Supplementary Supplementary Supplementary

J23100-ALeaderT-mRFP1

pSB1C3 pSB1K3 14 12 10 8 Expression level Expression 6 4 2

-7 -6 -5 -4 -3 -2 -1

log10 Kana (mol/L) Supplementary Supplementary

J23100-X-mRFP1

9 Aleader AleaderT 8 7 6 5 New-born RFP (A.U.) New-born 4 3

0 20 40 60 80 100

Time Supplementary Supplementary Supplementary Supplementary Supplementary

Test of Spinach with DFHBI 2.0 1.8 1.6 1.4 1.2 Fluorescence/OD 1.0 0.8 0.6

Csy4-loci Csy4-loci [+DFHBI] TRNA TRNA [+DFHBI]