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Championship Poster A riboswitch-controlled location-based networking framework Ezio Fok, Gloria Hlongwane, Natasia Kruger, Brad Marques and Sasha Reznichenko University of the Witwatersrand, Johannesburg, South Africa CHEMOTAXIS INTRODUCTION CHARACTERIZATION MOTILITY K537011 3.8 mm we performed a capillary The aim was to develop a biological communication network in which data is transferred as a s s a y t o s h o w t h a t E.coli CheZ deletion mutants FLUOROMETRY transformed E. coli CheZ Capillary Assay (Chemotaxis) chemical signals between bacterial populations. Any network requires connections between that were transformed with deletion mutants could to the nodes of the network. By engineering bacterial chemotaxis, the bacteria themselves can CheZ-Venus (under riboswitch Holding platform chemotactically migrate control) were inoculated in the form the network connections through their ability to locate and transmit messages between Theophylline riboswitch 1 CheZ Venus We were able to extensively towards theophylline. characterize fluorescent activation of centre of 85 mm semi-solid Riboswitch 1 CheZ Venus (K537011) PBS different bacterial populations. K537001 and K537011 Theophylline- agar plates containing 1.0 mM containing Venus associated with both Here we show that relative capillary tube containing 30 8 fold theophylline riboswitch 1 (K537001, theophylline capillary tube to Riboswitch 1 Venus e E.coli CHEMOTAXIS ) 25 K 5 3 7 0 11 ) a n d t h e o p h y l l i n e c (K537009) and Riboswitch n m There is evidence of movement e riboswitch 2 (K537002, K537012). n Parafilm Transformed baterial 2 Ve n u s K 5 3 7 0 1 0 ( ) K537011 c 20 8 culture droplet s of the bacteria from the point of K537012 2 e controls, Riboswitch 1 r CheB CheB 5 u inoculation. This can be t K537011 showed a 46-fold activation CheZ 15 Chemotaxis Index o 22.8 mm CheZ Venus K537011 ( ) a l f observed as a faint halo at 1.5 mM theophylline, whilst cfu of TEST CI = x100 U n 10 and Riboswitch 2 CheZ cfu of CONTROL CheW CheA F CheY CheW CheA a K537011 showed only a modest 8- surrounding the point of R CheW e CheA CheW CheA CheY * Venus (K537012) was able ( M inoculation. 5 fold activation. t o i n d u c e n o t e d Bacterial motility: Bacterial motility: Plate on Plate on CheR Tumble CheR 0 chemotactic behavior (as LB Agar LB Agar Riboswitch-CheZ Riboswitch-CheZ Run + Tumble K537012 showed ~ 5 fold 0 min 105 min These theophylline riboswitches are m e a s u r e d b y t h e improvements on previously- increase in motility compared to No analyte Riboswitch 2 CheZ Venus (K537012) Chemotaxis Index). + Analyte pSB1AC3 (- control) s u b m i t t e d r i b o s w i t c h e s b y K537011. Moreover, the CheZ- Riboswitch 1 CheZ Venus (K537011) CheZ Venus (K537013) (+ control) Venus fusion did not impair Lethbridge 2009 (K249028) and Riboswitch 1 Riboswitch 2 Riboswitch 1 Venus (K537009) NYMU Taipei 2010 (K411001). motility. Riboswitch 2 Venus (K537010) * P < 0.05 * P < 0.05 Bacterial chemotaxis is controlled via a signalling cascade, where CheZ helps 300 Riboswitch 1 CheZ Venus (K537011) 300 300 * x x Riboswitch 2 CheZ Venus (K537012) Riboswitch 2 CheZ Venus (K537012) e e d 250 direct movement of bacteria towards a stimulus. Riboswitch 1 Venus (K537009) 250 * d n ) Theophylline riboswitch 2 CheZ Venus i n Riboswitch 2 Venus (K537010) i m (RBS) CheZ Venus (K537013) 200 200 s 30 i CheZ Venus (K537013) s m i x NS K537002 and K537012 ( x NS a 150 150 t a d K537012 t 25 o e * * l o Our focus was to manipulate this mechanism to direct bacterial chemotaxis l 100 30 46 fold * 100 m e m e v 20 e h 50 a 50 h between two locations. This would enable motility towards a defined substance r C e t ) C c 25 15 e 0 0 n K537011 m K537012 c e followed by returning to the start location. n s s s n * P < 0.001 s s s u u u u u u c Riboswitch 1 CheZ Venus (K537011) 10 20 a n n n Riboswitch 1 CheZ Venus (K537011) n n n Riboswitch 2 CheZ Venus (K537012) t 8 e e e K537012 s e e e s V V V V V V 2 i e Z 2 Z r * Z 1 Z d 5 5 K537011 e e e e h * * h u * Riboswitch 1 Venus (K537009) h h h c h t c n t 15 C it C C i C o a a w l w 2 1 s Since riboswitches can be engineered for many substances, “Biotweet” is a f 0 s e o h o h c U b tc ib t m n i i 10 i R F R w w a 0 0.5 1.0 1.5 s s generic framework that may serve as the basis for the functioning of complex o o R e b ib ( i Theophylline (mM) R R biological communication networks and may be useful in various applications, M 5 including those of the medical and industrial sectors. 0 (RBS) CheZ Venus (K537013) 0 min 135 min pSB1AC3 (- control) Riboswitch 2 CheZ Venus (K537012) CheZ Venus (K537013) (+ control) OUTREACH GOALS Two riboswitches were used to control the translation of a CheZ-fluorescent EPIFLUORESCENCE MICROSCOPY protein, the first sensitive to theophylline and the second to atrazine. We identified sequence flaws in theophylline-sensitive riboswitches submitted K537009 K537010 Both theophylline riboswitch 1 to the registry (K249028, K411001) which may explain their poor ability to BF Venus BF Venus (K537003, K537009) and theophylline activate translation of downstream coding regions. We therefore aimed to riboswitch 2 (K537004, K537010) were able to upregulate Venus expression s improve on the riboswitches generated by previous iGEM teams s u u 0 mM upon theophylline induction. n n e e ) Theophylline (K537001,K53011). ) V V 0 9 1 0 2 1 0 K537010 showed robust activation with 0 7 h 7 h c 3 c 3 some evidence of “leaky” expression at t t i 5 i A new theophylline-sensitive riboswitches generated from the Gallivan lab 5 Outreach Survey w K w K baseline. ( s ( s The team developed an online survey which was distributed o appears to have much better properties (our entries: K537002, K537012). We o 1.5 mM Problem identification: b b i i amongst different individuals, mostly students. The survey was R Theophylline tested this riboswitch’s ability to activate expression of a CheZ-Venus reporter R Venus activation not as robust as CheZ- focused on determining what people knew about synthetic biology fusion, and to test this system using motility and chemotaxis assays. Venus fusions for both riboswitches, yet 1) Many South African students come from resource-poor bot riboswitches are functionally communities, and are less inclined to take subjects such as and concepts such as genetic engineering, and how they felt about it Riboswitch 1 Venus (K537009) Riboswitch 2 Venus (K537010) induced and represent improvements on mathematics and science due to the perceived difficulties, as a technology. parts: K249028 and K411001. poor role models and a lack of career guidance. Sci-Bono Science Week 2) Synthetic biology is also a relatively unknown concept in South Sci-Bono is an organisation that strives to promote a love of science Africa, especially amongst students, not only from primary and amongst children and youth. The team approached the organisation ACHIEVEMENTS AT EUROPEAN high schools, but even amongst those at university level. and requested that they present to underprivileged children during National Science Week. Large groups of schoolchildren attended MEET THE TEAM REGIONAL JAMBOREE: By participating in workshops and interactive discussions, the the team's presentations during that week. The presentations were BIOTWEET SIMULATOR Wits_CSIR_SA 2011 team gained insight into the level of divided into one aimed at primary school children, which was about awareness regarding synthetic biology amongst the South scientific discoveries and how science can be cool; and a workshop Regional Finalist Africans learners from resource poor/underprivileged for high school children which introduced them to synthetic biology backgrounds. We feel we gained invaluable knowledge of the by getting them to design their own 'superhero' bacteria. The team Best Experimental Measurement difficulties and challenges facing the field of synthetic biology, and also manned a stand, along with team advisor Laura Millroy, which Best Poster in implementing technologies such as “biotweet” in this contextual gave children information about career options and what subjects to environment. take at school to get into science courses at university. IMPROVEMENTS SINCE THE EUROPEANREGIONAL JAMBOREE: SPONSORS Used a different mounting technique that enabled us to improve our epi- fluorescence microscopy images At START (Atrazine) At Theophylline Performed key experiments in triplicate Non-responsive to Atrazine Non-responsive to and showed statistical significance for our Theophylline The simulation tool models the chemotactic motion of bacteria on a petri Responsive to Theophylline Responsive to Atrazine Team members from left to right. Gloria, best parts dish. Two chemo-attractants (shown in red and green) are present in the Theophylline activates motility Switch responsiveness to Ezio, Sasha, Natasia and Brad (with on a concentration gradient Atrazine (by activation of Cre environment and influence the motile action of the bacteria. Various Improved the datasheets, registry entries caused by presence of IPTG); options are available to the user of the tool to specify the experiment Johannesburg’s Hillbrow Tower in the and overall aesthetics of our wiki Response to Atrazine is The team wishes to additionally acknowledge the support from advisors and supervisors: Ms Michelle Robinson, Prof Marco activated AND response to setup.The simulator was developed in the C# language and utilises the background) Theophylline is switched off Unity3D graphics engine.
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