Online Supplementary information for: A modular RNA interference system for multiplexed gene regulation Ari Dwijayanti1,2, Marko Storch1,2,3, Guy-Bart Stan2,4*, Geoff S. Baldwin1,2* 1 Department of Life Sciences, Imperial College London, London SW7 2AZ, UK 2 Imperial College Centre for Synthetic Biology, Imperial College London, London SW7 2AZ, UK 3 London Biofoundry, Imperial College Translation & Innovation Hub, London W12 0BZ, UK 4 Department of Bioengineering, Imperial College London, London SW7 2AZ, United Kingdom * To whom correspondence should be addressed. Email: [email protected]; [email protected] Supplementary Fig. 1. Predicted base-pairing interactions of the designed mARi and mRNA target. Supplementary Fig. 2. Characterisation of constitutive promoters and transcript stoichiometry. Supplementary Fig. 3. Growth profile of different host strains expressing the mARi-based repression system. Supplementary Fig. 4. mARi-based regulation in different E. coli strains. Supplementary Fig. 5. Orthogonal repression by mARi. Supplementary Fig. 6. Multiplexed and simultaneous gene regulation by mARi. Supplementary Fig. 7. Maps for the plasmids used in this study. Supplementary Table 1. Summary of mARi variants used to evaluate the impact of the position of the target site. Supplementary Table 2. UTR-RBS BASIC linker sequences used for testing the performance of mARi-A when combined with different RBS. Supplementary Table 3. The calculated relative expression ratio and repression activity of mARi. Supplementary Table 4. Orthogonal mARis and off-target prediction towards E. coli genome. Supplementary Table 5. List of standardised bioparts sequences used in this study. Supplementary Table 6. List of orthogonal BASIC linker sequences used in this study. UTR -A -RBSc a +1 5’ RiboJ RBS ATG..(sfGFP) 3’ Position 1 +82 +115 Position 2 +102 +127 Position 3 +111 +135 Position 4 +133 +155 b 1 Target (sfGFP) n o i t i s o P mARi 2 Target (sfGFP) n o i t i s o P mARi 3 Target (sfGFP) n o i t i s o P mARi 4 n o Target (sfGFP) i t i s o P mARi Supplementary Fig. 1. Predicted base-pairing interactions of the designed mARi and mRNA target. (a) Schematic design of target site selection (Positions 1-4) of mARi-mediated repression. Arrows show the direction of the reverse complementary sequence in the mARi design. (b) Predicted base pairing of mARi-A with mRNA targets (sfGFP) for the four different target positions. Numbers indicate the relative positions of bases from the Transcription Start Site (+1). a b Promoter mARi Promoter mRNA UTR -A- 27154 30000 RBSc ) u ) a ( u a y p15A T T t i , e s n c n 5x a n e t e 20000 sfGFP e n i c m P J23xxx_BASIC s e c e i c r r n t o 11619 e e u c l s m F e o 8242 r 10000 e o u g l ( F 2705 1362 0 J23119 J23111 J23104 J23101 J23108 PJ23xxx_BASIC Promoter mRNA (P ) c J23xxx_BASIC J23111_BASIC J23104_BASIC J23101_BASIC J23108_BASIC Without ARi 1.2 With ARi e c n e 1.0 c e s c e r n o e u l c f s 0.8 n e r a o e u m l f c i d r t 0.6 e e s i l m o a e m g r 0.4 o d e N z i l a m r o 0.2 N 0.0 l l l l 9 1 4 1 9 1 4 1 9 1 4 1 9 1 4 1 o o o o r 1 1 0 0 r 1 1 0 0 r 1 1 0 0 r 1 1 0 0 t t t t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 n n n n 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 o o o o 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 c c c c J J J J J J J J J J J J J J J J Promoter mARi (P ) J23xxx_BASIC Supplementary Fig. 2. Characterisation of constitutive promoters and transcript stoichiometry. (a) Schematic of the expression system used to characterise our standardised constitutive promoter set. (b) Promoter activity for the set of the standardised constitutive promoters used for driving mARi and mRNA expression. This promoter activity was used to calculate the relative expression ratio (mARi:mRNA) in Fig. 3a. The subsets of promoters used for mRNA and mARi are indicated by the coloured wedges. (c) Normalised fluorescence from flow cytometry assay for all constructs used to evaluate transcript stoichiometry in Fig. 3c. Data are shown as the mean ± SD of three biological repeats (black dots). The calculated relative expression ratio and repression activity of mARi are provided in Supplementary Table 3. Supplementary Fig. 3. Growth profile of different host strains expressing the mARi-based repression system. Growth curves of strains with and without mARi expression in different host strains from a plate reader assay: (a) DH5α (K-12 strain), (b) DH10b (K-12 strain), (c) BL21(DE3) (B strain) and (d) BL21star(DE3) (B strain). Data associated with Fig. 3g were taken during early stationary phase (blue dotted line); Data associated with Fig. 3h were taken during mid-exponential phase at around 4h (orange dotted line) and early stationary phase at around 8h (blue dotted line). Lines show the mean from 3 independent measurements with the shaded area showing ± SD. Statistically significant differences were determined using Student’s t-test (ns used to denote “not significant”). DH5α DH10b BL21(DE3) BL21star(DE3) 1.4 Without mARi With mARi (high ratio) 1.2 e c 0 n 0 1.0 e 6 c s s b e A r / l o 0.8 F u l f d e d z e i l 0.6 s i a l a m r m o r 0.4 o N N 0.2 0.0 1 A 1 1 A 1 1 A 1 1 A 1 0 5 B 0 5 B 0 5 B 0 5 B 1 1 1 1 1 1 1 1 p M p M p M p M SC p SC p SC p SC p p p p p Supplementary Fig. 4. mARi-based regulation in different E. coli strains. The performance of mARi repression with a high expression ratio (mARi>mRNA), single plasmid system in four host strains was measured using a plate reader assay. Strains DH5α, DH10b, BL21(DE3), and BL21star(DE3) were used, with three different plasmid copy numbers: pSC101, p15A, and pMB1. Data were taken during early stationary phase (8 h). Data associated with Fig. 3g, h were taken from the p15A backbone. Data are shown as the mean ± SD of three independent repeats (black dots). a Target sequence b UTR -A UTR -B UTR -C UTR -D UTR -E 100 UTR -A ) % ( y pMB1 T p15A T T t i e t c n UTR -X n UTR -B e e d mARi-X -RBSc I u q e s t UTR -C e 0 g r a T UTR -D UTR -E c UTR- A UTR -B UTR -C UTR -D UTR-E 2.0 Without mARi e c With mARi n **** **** e 1.8 **** ** **** c s e r 1.6 o u l f 1.4 n a e 1.2 m c i r 1.0 t e m 0.8 o e g 0.6 d e z i l 0.4 a m r o 0.2 N 0.0 l l l l l A B E A B E A B E A B E A B E C D C D C D C D C D o o o o o - - - - - - - - - - - - - - - - - - - - - - - - - r r r r r i i i i i i i i i i i i i i i i i i i i i i i i i t t t t t R R R R R R R R R R R R R R R n n n n n R R R R R R R R R R o o o o o A A A A A A A A A A A A A A A A A A A A A A A A A c c c c c m m m m m m m m m m m m m m m m m m m m m m m m m Supplementary Fig. 5. Orthogonal repression by mARi. (a) The calculated identity similarities of each target sequence in UTR pairs was obtained using the EMBOSS needle method (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) (b) Schematic of the genetic constructs used to evaluate target specificity of modular mARi-X and UTR-X pairs. The mRNA expression cassettes driven by PJ23101_BASIC were located in the p15A backbone while the mARi expression cassettes with PJ23119 were cloned in a pMB1 backbone. Both expression plasmids were then co-transformed into DH5α cells. (c) Response of all mARi and target sequence combinations were measured by flow cytometry assay after 6 h of incubation, and the fluorescence was normalised to control cells with sfGFP and without mARi. Data are shown as the mean ± SD of three independent repeats (black dots). Statistically significant differences were determined using Student’s t-test (**** represents p<0.0001, ** represents p<0.01).