Team:BOKU-Vienna/Design

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Project Inspiration Design

Design of the Switches

An aptamer-inducible switch as the key concept of the test

In specific, and as opposed to the existing approaches, our idea includes the genetic modification of the Escherichia coli strain DH10B to engineer it into generating a specific signal in the presence of Mycobacterium ulcerans. The signal shall be based on a genetic switch which is capable of detecting mycolactone, by specific bonding to an aptamer sequence ^[3, ^6]. Upon presence of mycolactone, a colour reaction shall be induced in the test microorganism to produce a swift and clear response. A signal enhancer shall be incorporated into the circuit to improve the limit of detection. A default signal for cell viability control is realized with

Figure 1: Schematic representation of our concept

Molecular design of the bio-sensor

The cornerstone of the switch is the riboswitch sequence, where we have a variety of structures available to be screened for the best fit for our purpose. In the default stage, upon DNA transcription, the Aptamer sequence binds to the terminator sequence forming a hairpin structure ^[4, ^7]. A protein on the RNA polymerase binds to the hairpin which stops the transcription. The Uracil stretch prevents strong binding of the RNA polymerase to the mRNA and causes its dissociation from the mRNA. The genetic toggle switch stays in its default mode. If mycolactone is present, however, the hairpin structure is opened up, preventing the fixation of the RNA polymerase by the hairpin structure. The RNA polymerase transcribes the signal molecule mRNA. Consequently, the aptamer switch is capable of activating a cascade of signals. We have decided to test two different circuit designs with several modifications each, so as to allow us to screen for the most efficient option.

Two circuit design propositions for output signal generation

In the first circuit version, the mycolactone bound to the aptamer induces the expression of LuxI ^[9]. Together with the constitutively expressed LuxR ^[10], a complex is formed which induces the Lux promotor ^[11]. Under the control of this promotor is a reporter blue chromoprotein (amilCP) andan additional LuxI for enhancement ^[12]. The signal is amplified by N-Acyl homoserine lactone(AHL), a molecule produced by LuxI. AHL is released into the environment and received by other cells where it activates the Lux Promoter. For viability control a green fluorescent protein (GFP) is under the control of a LacI/T7 Promoter. By adding lactose into the medium GFP will be expressed and a clear distinction of the default and positive signal will be possible. (Figure 2)

Figure 2: Version 1 of the genetic expression system

The other version is based on the expression of T7-Polymerase upon mycolactone binding to the aptamer ^[13]. This, in turn, induces expression of LuxI which forms a complex with the constitutively expressed LuxR. This complex activates the Lux Promoter which transcribes the blue Chromoprotein (amilCP). The signal amplification works the same as in version 1. For viability control this expression system contains an Arabinose Promoter. By adding Arabinose to the medium, the transcription of the green fluorescent protein will be induced. (Figure 3).

Figure 3: Version 2 of the genetic expression system

Additional system without Quorum Sensing

Additionally, we decided to try to build a System without the quorum sensing part, in case something wouldn’t work, or the time would be too limiting to build the entire system. Which turned out to be the right decision. During the months in the Lab we faced several problems with different Parts of the quorum sensing system. But still, our system worked very well with the T7-Polymerase being induced by the right Riboswitch-design and further inducing the amilCP trough a T7-Promtor.We also added the arabinose induced GFP viability signal to that design (Figure 4). You can read more about our actual “Results”.

Figure 4: amilCP directly induced by T7 Promoter after the binding of the T7-Polymerase to the Riboswitch. For viability control GFP production is induced by the arabinose promoter.

The anticipated advantages of the novel diagnostic approach

As can be seen from the proposed designs, the switch is highly specific for mycolactone as its induction depends on the binding of mycolactone to a specific aptamer sequence ^[4,5,8]. Moreover, the signal which is generated is a simple visual read out which sets the method apart from previously suggested versions such as LAMP or thin layer chromatography. Most importantly, however, the detection limit may be lowered significantly, as the enhancers and upregulation basically allow for a production of chromoprotein upon the exposure to a low number of mycolactone molecules. In further steps, should there still be time, we hope to include a more elaborate upregulation and enhancement mechanism, so that the presence of one molecule of mycolactone and the binding to one aptamer in one cell will eventually lead to readable signal output. The design of the latter, however, strongly depends on the choice of designs described above, and will thus be detailed at a later stage. For now, we are looking forward to starting our project and hope to inspire more people to use and apply synthetic biology as solutions for current problems in various contexts.

Links

[1] WHO, 2019: Fact Sheet on Buruli Ulcer.

[2] WHO, 2016: Distribution of Buruli Ulcer worldwide.

[3] Sakyi, .; Aboagye, S.; Otchere, I.D. and Yeboah-Manu, D. (2016). Clinical and Laboratory Diagnosis of Buruli Ulcer Disease: A Systematic Review. Canadian Journal of Infectious Diseases and Medical Microbiology. Vol. 2016.

[4] Sakyi, S.A.; Aboagye, S.Y.; Otchere, I.D.; Liao, A.M.; Caltagirone, T.G. and Yeboah-Manu, D. (2016) RNA Aptamer That Specifically Binds to Mycolactone and Serves as a Diagnostic Tool for Diagnosis of Buruli Ulcer. PLoS Negl Trop Dis. Volume 10(10).

[5] Berens, C.; Groher, F. and Suess, D. (2014). RNA aptamers as genetic control devices: The potential of riboswitches as synthetic elements for regulating gene expression. Biotechnol J. Volume 10(2). doi: 10.1002/biot.201300498

[6] Zhang, J.; Lau, M.W. and Ferré-D’Amaré, A.R. (2010). Ribozymes and Riboswitches: Modulation of RNA function by small molecules. Biochemistry. Volume 49(43). doi: 10.1021/bi1012645.

[7] Tobias, N. J. ; Seemann, T.; Pidot, S. J.; Porter, J. L.; Marsollier, L.; Marion, E. and Stinear, T. P. (2009). Mycolactone gene expression is controlled by strong SigA-like promoters with utility in studies of Mycobacterium ulcerans and buruli ulcer. PLoS neglected tropical diseases. Volume 3(11)

[8] Wadagni, A.M.; Frimpong, D.M.; Phanzu, A.; Ablordey, E.; Kacou, M.; Gbedevi, E.; Marion, E.; Xing, Y.; Babu, V.S.; Phillips, R.O.; Wansbrough-Jones, M.; Kishi, Y. and Asiedu, K. (2015). Simple, Rapid Mycobacterium ulcerans Disease Diagnosis from Clinical Samples by Fluorescence of Mycolactone on Thin Layer Chromatography. PLoS Neglected Tropical Diseases. Volume 9(11). doi:10.1371/journal.pntd.0004247

[9] Braff, J.C. (2004). Lux I: BBa_C0161. Group: Antiquity.

[10] Mahajan, V.S.; Marinescu, V.D.; Chow, B.; Wissner-Gross, A. and Carr, P. (2003). LuxR for AHL binding: BBa_C0062. Group: Antiquity.

[11] Mahajan, V.S.; Marinescu, V.D.; Chow, B.; Wissner-Gross, A. and Carr, P. (2003). LuxR + AHL inducible promoter lux pR: Part: BBa_R0062. Group: Antiquity

[12] Sun, L. (2011). Parts BBa_K592009 and BBa_K592010. Team iGEM11_Uppsala-Sweden

[13] Gao, C. (2015). Part BBa_K1706007. Group: iGEM15_Jilin_China

[14] Gaber, R. (2007). Part BBa_I712074. Group iGEM07__Ljubljana