Team:SDU-Denmark/Model

Project Design









Dormancy System

Project Overview


Introduction


Cyanobacteria contain signal transduction systems, thereby making them capable of sensing and responding to light Bussell AN, Kehoe DM. Control of a four-color sensing photoreceptor by a two-color sensing photoreceptor reveals complex light regulation in cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(31):12834-9.. This ability gives the organisms the opportunity to adapt and optimize their metabolism to a circadian rhythm. Photoreceptors in the plasma membrane, of which phytochromes are especially abundant and well described, are responsible for this property Vierstra RD, Davis SJ. Bacteriophytochromes: new tools for understanding phytochrome signal transduction. Seminars in cell & developmental biology. 2000;11(6):511-21.. In 2004, the UT Austin iGEM team made a light response system consisting of a photoreceptor combined with an intracellular indigenous regulator system Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, et al. Synthetic biology: engineering Escherichia coli to see light. Nature. 2005;438(7067):441-2.. EnvZ and OmpR make up the two-component system naturally found in E. coli. The photoreceptor known as Cph1 was isolated from the cyanobacteria Synechocytis PCC6803. Cph1 has functional combination sites, which combined with the kinase EnvZ form a two-domain receptor, known as Cph8. Activation of Cph8 is mediated by the chromophore phycocyanobilin, PCB, that is sensitive to red light with maximal absorbance at 662 nm Lamparter T, Esteban B, Hughes J. Phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803. Purification, assembly, and quaternary structure. European journal of biochemistry. 2001;268(17):4720-30..
When not exposed to light, PCB activates the phytochrome Cph1, thus promoting kinase activity through the EnvZ kinase. When the transcription factor OmpR is phosphorylated by EnvZ, expression of genes regulated by the OmpR-regulated promoter is initiated. Excitation of PCB by red light results in a situation where the transcription factor OmpR is not regulated. The absence of phosphorylated OmpR leads to no activation of the OmpR-regulated promoter, thereby preventing gene expression.



Figure 1. Left: Red light activates PCB, which in turn inactivates the photoreceptor complex Cph8, preventing gene expression from the OmpR-regulated promoter. Right: In absence of light, PCB is inactive, which enables the Cph8 to phosphorylate the transcription factor OmpR. This promotes gene expression from the OmpR-regulated promoter.


The photocontrol device can be used to regulate a toxin-antitoxin system, enabling the implementation of a light-dependent dormancy system. A toxin-antitoxin system is composed of two gene products, a cytotoxin and an antitoxin, the latter which neutralises the the toxic effect caused by the toxin. In E. coli K-12 the cytotoxin RelE and antitoxin RelB comprise such a system Gotfredsen M, Gerdes K. The Escherichia coli relBE genes belong to a new toxin-antitoxin gene family. Molecular microbiology. 1998;29(4):1065-76.. Expression of the cytotoxin RelE inhibits translation in the cells, due to its ability to cleave mRNA found in the A-site of the ribosome. RelB neutralises the toxic effect of RelE through interaction between the two proteins. Whether the cell lies dormant in response to expression of RelE depends on the ratio of antitoxin RelB and RelE present in the cell. Several studies have shown that RelB and RelE form a complex with RelB:RelE stoichiometry of 2:1 Overgaard M, Borch J, Gerdes K. RelB and RelE of Escherichia coli form a tight complex that represses transcription via the ribbon-helix-helix motif in RelB. Journal of molecular biology. 2009;394(2):183-96.Overgaard M, Borch J, Jorgensen MG, Gerdes K. Messenger RNA interferase RelE controls relBE transcription by conditional cooperativity. Molecular microbiology. 2008;69(4):841-57.. When the RelB:RelE stoichiometric-ratio is lowered to 1:1, studies show that RelB is not able to protect the cells against the RelE-caused translational inhibition Overgaard M, Borch J, Jorgensen MG, Gerdes K. Messenger RNA interferase RelE controls relBE transcription by conditional cooperativity. Molecular microbiology. 2008;69(4):841-57.. For further information about the light-dependent dormancy system, read here.


Modelling



Modelling of the RelE-RelB System is Essential to Avoid Irrevocable Dormancy

Controllable dormancy is a feature that holds the potential to be applied in many different situations. However, inducing dormancy and bringing the bacteria back to a metabolic active state is like balancing on a tightrope, and to establish the basis of future implementations, the properties of this system would have to be investigated further. In an endeavour to provide this basic knowledge, stochastic modelling utilising the Gillespie algorithm was performed in an attempt to prognosticate the system and simulate the interactions between the toxin and antitoxin. The toxin RelE is inhibited by the antitoxin RelB through complex formation, and both proteins interact with their promoter in a feedback mechanism. To consolidate the model, the capacity of the toxin-antitoxin system was assessed in an experiment, as the controllability of the dormancy system was studied through manual regulation of RelE and RelB expression.
You can read more about the modelling here.



Figure 2. Left: The time required for the bacteria to enter dormancy varies with the expression level of RelB. The percentage of dormant bacteria, defined as containing RelE amounts above 40 molecules per cell as a function of time in minutes. Right: Only one of the tested configurations, RelB2:50-RelE:35, causes the bacteria to regain their activity within the modelled time. The percentage of dormant bacteria, defined as containing RelE amounts above 15 molecules per cell as a function of time in minutes. The data is based on the simulation of 1000 independent bacteria.

The simulated data revealed, that when enhanced RelE production is implemented, in order to induce dormancy in E. coli, the effect come easily. However, implementation of RelB expression is also found necessary to ensure that the bacteria are able to enter an active state again.
The model showed that the system is sensitive to the RelE:RelB ratio, as well as the total amount of produced toxin. As seen in Figure 2, implementation with production rates in the vicinity of 50 and 35 molecules per minute for RelB and RelE respectively, was found to be suitable for balancing our system; the bacteria lay dormant within the computed time and re-enter an active state within minutes.
The simulated data made it evident that implementing an optimised dormancy system comprises a challenge, as the individual expression levels of RelE and RelB, as well as their interaction, has a crucial impact on the regulation of dormancy. Thus, controlled gene expression of both RelE and RelB is required to implement a controllable dormancy system in the PowerLeaf.
If you want to dig deeper into this crucial modelling of the dormancy system, read the full results here.

Approach


In 2004 the Austen and UCSF iGEM team created a device sensitive to light, laying the foundation for the Coliroid project. In this project, the system is combined with the RelE-RelB toxin-antitoxin system in the endeavour to mediate light-dependent dormancy in bacteria. As tight regulation is required for the RelE-RelB system Tashiro Y, Kawata K, Taniuchi A, Kakinuma K, May T, Okabe S. RelE-Mediated Dormancy Is Enhanced at High Cell Density in Escherichia coli. J Bacteriol. 2012;194(5):1169-76., modelling of the toxin-antitoxin system is essential. The impact of different RelE-RelB expression levels was simulated by modelling. Using the results obtained by this modelling, a hypothetical working system-design was devised.
On basis of the modulated system, the potential of different vectors and promoters in various combinations was tested. This constitutes the foundation for how the design of the light-dependent dormancy system in E. coli has been optimised, and the final approach shaped. Ultimately, the light-dependent dormancy system, which is illustrated in Figure 3, was composed of the following parts:

  • The photocontrol device controlled by the PenI-regulated promoter, BBa_R0074, on a high copy vector.
  • The antitoxin RelB controlled by pBAD, BBa_K2449031, on a low copy vector.
  • The toxin RelE controlled by the OmpR-regulated promoter, BBa_R0082, on either a low copy vector or the chromosome.

For further information about our approach, read here.



Figure 3. The final design of the light-dependent dormancy system. RelE under control of the OmpR-regulated promoter on the chromosome or a low copy vector, RelB under the control of the pBAD promoter on a low copy vector, and the photocontrol device controlled by the PenI-regulated promoter on a high copy vector.