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.

RelE and RelB Regulate Dormancy and Influence Their Own Expression

Modelling of the effects of different RelE and RelB expression levels were performed as an important aspect in the implementation of the RelE-RelB toxin-antitoxin system. The toxin RelE constrains bacterial growth by mRNA degradation, thereby inhibiting translation, whereas the antitoxin RelB inhibits this toxic effect by forming complexes with RelE. As seen in Figure 1, three different protein complexes are formed, namely RelB₂, RelB₂RelE, and RelB₂RelE₂, containing zero, one, and two RelE molecules respectively Guang-Yao Li, Yonglong Zhang, Masayori Inouye, Mitsuhiko Ikura, Structural Mechanism of Transcriptional Autorepression of the Escherichia coli RelB/RelE Antitoxin/Toxin Module, In Journal of Molecular Biology, Volume 380, Issue 1, 2008, Pages 107-119, ISSN 0022-2836.

Figure 1. The three toxin-antitoxin complexes RelB₂, RelB₂RelE, and RelB₂RelE₂.

The expression of both RelE and RelB is regulated by the relBE promoter, which is influenced differently by each of the complexes, as seen in Figure 2. When small amounts of RelE is present, RelB₂ and RelB₂RelE repress transcription through relBE by binding to the operator sequence. However, when high amounts of RelE are present, the toxin mitigates this repression by reacting with complexes bound to the operator sequence Cataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297.

Figure 2. The interactions between the toxin-antitoxin complexes and the relBE promoter controlling the expression of RelE and RelB. RelE mediates the degradation of mRNA, thereby inhibiting translation.

During starvation, the half-life of RelB decreases significantly due to a Lon-proteaseOvergaard, M., Borch, J., Jørgensen, M. G. and Gerdes, K. (2008), Messenger RNA interferase RelE controls relBE transcription by conditional cooperativity. Molecular Microbiology, 69: 841–857. doi:10.1111/j.1365-2958.2008.06313.x, causing a shift in the equilibrium of RelB and RelE to a higher level of RelE. In a non-starvation situation, the interactions with the operator sequence keeps the amount of free RelE at a low level, thereby stabilising the system Cataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297. In our simulation, the shift in equilibrium is made by introducing additional expression of RelE.
Two different models were used with two different approaches:

How a given configuration of RelB and RelE production increases the RelE concentration and whether it could induce dormancy within 2 hours.
The time required for the bacteria to exit dormancy for each of the configurations, that is, how long it takes the levels of free RelE to decrease again.

Rates and Reactions

Molecule	Initial number of copies
mRNA	7
RelB₂	410
RelE	0
RelB₂RelE	65
RelB₂RelE₂	11
Free operator sites	0
O(RelB₂)	2
O(RelB₂RelE)	0
O(RelB₂RelE)₂	2

All values are integers as the Gillespie algorithm works with discrete numbers of molecules. The values were chosen based on a stable equilibrium found by letting the model run a simulation of the inherent system over 450 minutes with different starting values.
Ranging from 1-350 molecules per minute, the implemented expression rates of RelE and RelB in the model might seem too high, as the rates in the inherit system is effectively around 80-100 for RelB and 2-5 for RelE. However, the possibility of placing the relE and relB genes under regulation of controllable promoters makes the high total production values reasonable.
When the inherent toxin-antitoxin system is activated under starvation, 40-70 molecules of free RelE are found in each cell, making it reasonable to believe that the cells enter dormancy when a few tens of free RelE copies are present. This result obtained from the model is in agreement with literatureCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297
For the full list of constants, see the attached Table 1 at the bottom of this page.

Running the Model

To give a stochastic view of the system, the Gillespie algorithm was run in the computer programming language MATLAB, utilising an implementation made by MATLAB user Nezar.
For the runs simulating dormancy, deterministic initial values were used and the system was run for 30 minutes without activation of the inserted toxin promoter. This resulted in a stochastic distribution of initial values mimicking variations between cells. Analysis showed, that 30 minutes was enough for the model to find a stable distribution, which is realistic considering the growth cycle of an E. coli cell.
For the runs simulating activation, the data generated at the end of a dormancy run was used as initial value and deactivated expression of RelE. When the concentration of free RelE decreases to below 15 copies, a cell is considered active. This value was probably set too low, but tests displayed marginal difference between 15 and 45 copies, where the lower limit was chosen to decrease uncertainty of the cell state.
All runs simulated 1000 cells, which should be sufficient to get stable averages and the model assumed well-mixed conditions in every cell and considered each cell independently. Furthermore, the model has no cut-off for maximum values of RelE, as the exact relation between RelE concentration and dormancy state is unknown, yet a functional cut-off was found through activation times.
Download of Matlab-scripts

Table 1

Constant	Identifier	Units	Value
mRNA transcription rateCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297	α₀	1/min	154.665
mRNA half-lifeCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297	τ_m	min	7.2
RelB half-lifeCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297Overgaard 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. J. Mol. Biol. 2009;394:183–196. doi: 10.1016/j.jmb.2009.09.006	τ_B	min	4.3
RelE half-lifeCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297	τ_E	min	43 (growing) 2000 (dormant)
Bound RelB half-lifeCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297Overgaard 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. J. Mol. Biol. 2009;394:183–196. doi: 10.1016/j.jmb.2009.09.006	τ_c	min	17
RelB transcription rateCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297	trans_B	1/min	15
RelE transcription rateCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297	trans_E	1/min	0.3
Binding rateSneppen K, Zocchi G. Physics in Molecular Biology. Cambridge, UK: Cambridge University Press; 2005.	k _b	1/min	3.8
Dissociation rate B₂EOvergaard, M., Borch, J., Jørgensen, M. G. and Gerdes, K. (2008), Messenger RNA interferase RelE controls relBE transcription by conditional cooperativity. Molecular Microbiology, 69: 841–857. doi:10.1111/j.1365-2958.2008.06313.x	K_D (B₂E)	molecules	0.3
Dissociation rate B₂E₂Overgaard, M., Borch, J., Jørgensen, M. G. and Gerdes, K. (2008), Messenger RNA interferase RelE controls relBE transcription by conditional cooperativity. Molecular Microbiology, 69: 841–857. doi:10.1111/j.1365-2958.2008.06313.x	K_D(B₂E₂)	molecules	0.3
Dissociation rate O.B_fGotfredsen, M. and Gerdes, K. (1998), The Escherichia coli relBE genes belong to a new toxin–antitoxin gene family. Molecular Microbiology, 29: 1065–1076. doi:10.1046/j.1365-2958.1998.00993.xCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297	K_D1	molecules	10
Dissociation rate O.B₂EGotfredsen, M. and Gerdes, K. (1998), The Escherichia coli relBE genes belong to a new toxin–antitoxin gene family. Molecular Microbiology, 29: 1065–1076. doi:10.1046/j.1365-2958.1998.00993.xCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297	K_D2	molecules	0.04
Dissociation rate O.(B₂E)₂Gotfredsen, M. and Gerdes, K. (1998), The Escherichia coli relBE genes belong to a new toxin–antitoxin gene family. Molecular Microbiology, 29: 1065–1076. doi:10.1046/j.1365-2958.1998.00993.xCataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297	K_D3	molecules	30
Cleavage ratePedersen K, et al. The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. Cell. 2003;112:131–140. doi: 10.1016/S0092-8674(02)01248-5 Cataudella I., Trusina A., Sneppen K., Gerdes K., Mitarai N. Conditional cooperativity in toxin-antitoxin regulation prevents random toxin activation and promotes fast translational recovery. Nucleic Acids Res. 2012;40:6424–6434. doi: 10.1093/nar/gks297	k_c	1/min 1/molecules	2.0

Results

As the bacteria only require a few tens of RelE molecules to enter dormancy, the threshold was placed at 40 copies to allow for lag in activation. This is of course an oversimplification, but it is not a problem that activation and dormancy are defined at different levels, since the exact number of RelE molecules required to induce dormancy is unknown.

The Impact of Promoters with Different Production Rates on RelE Expression
During the implementation of RelE, gene expression was simulated for promoters with different strengths, which were chosen through an iterative process. Promoters with production rates of 3.5 and 10.5 molecules per minute, both induce dormancy rather slowly, with the latter inducing dormancy in approximately 50 minutes. In cells cloned with a promoter producing 35 molecules per minute, the cells will enter dormancy in about 10 minutes, while promoters producing 105 and 350 molecules per minute both have a negligible timeframe. In the simulated dormancy system, the three strongest promoters exhibited similar results, indicating that a certain threshold value had been transcended. This means, that not only was the gene expression disproportional to the promoter strength, but the risk of overshooting was also increased tremendously.

Figure 1. The increase of free RelE molecules in E. coli cells, after activation of the artificial RelE production, shown logarithmically. The condition for induced dormancy is an amount of free RelE molecules around tens of copies. The three highest levels of RelE production, correlating with the highest promoter strengths, show little difference in the time at which dormancy occurs. When the RelE production is set to 10.5 molecules per minute, dormancy is induced more slowly and stabilises at lower concentrations. The lowest RelE production value does not trigger dormancy, and has only little effect on the system.

RelB is Required for Activation of Bacteria after Dormant State
If RelB was not expressed, the bacteria remained dormant for hours after RelE production had ceased, making it clear, that production of the antitoxin RelB was necessary for activation. Considering the stability of RelE in non-growing conditions, it was not surprising to find that RelB production would be the primary element in sequestering free RelE.

Figure 2. The logarithmic plots shows that the decrease in free RelE in dormant bacteria is low without artificial expression of RelB. None of the simulated bacteria reentered an active state within the modelled time.

Appropriate Ratio of RelE and RelB Expression is Essential
Different expression rates of RelB were combined with production rates of RelE at 35 and 100 molecules per minute, corresponding to relatively medium and strong expression levels respectively. This revealed that variation in RelB had a higher impact on the time required before the dormant state was reached when the RelE production is lowered. Out of the established configurations, the RelB₂:50-RelE:35 configuration showed promising results. Compared to the RelB₂:35-RelE:35 configuration, where only few bacteria reenter an active state within the modelled time set to 2.5 hours, the RelB₂:50-RelE:35 configuration revealed a high sensitivity to the expression level of RelB. This indicated a need for the expression of RelB to be higher than RelE, but as the best results were achieved at low production rates of RelE, it is important to stringently control the expression of RelB to ensure that the bacteria are able to enter dormancy.

Figure 3. The variation in time required for the bacteria to enter an active state for different expression levels of RelB is dependent on the level of RelE expression. All configurations achieve dormancy within the modelled time.

Figure 4. RelB₂:50-RelE:35 induces an active state within minutes, whereas RelB:35-RelE:35 only causes few of the bacteria to enter an active state. In the remaining configurations all bacteria remained dormant.

Discussion of Model Regarding the Artificial Dormancy System

Model Limitations
As the model only simulates the dormancy for 2 hours, not all configurations have reached equilibrium, therefore these configurations might attain a higher concentration of free RelE than modelled. This could result in a prolonged phase reentering an active state. The activation model has a rather weak predictability, as the half-life of RelE is quite high. This essentially means, that the model is unable to reduce the total amount of RelE, given by RelE_tot=RelE_free+RelE_bound, because of the short simulated timespan. Thus, in activation runs, where the number of free RelE molecules reaches a high level, the amount of RelE_bound is equally high. The problem arises, since the model works under the assumption that bound RelB is not completely stable, causing RelE-RelB complexes to dissociate, whereby RelE is freed. This causes the induced RelB expression to approach equilibrium, implying that the decrease in free RelE is rather slow. It is therefore not only the amount of free RelE that determines the activation time, but also whether the amount of dissociating complexes is high enough to counter the RelB production. Hence, it has no relevance to further test high RelE production in this model, as a high number of complexes will easily be achieved.
Light Sensitivity
One thing the model does not include, is the actual sensitivity to light. Out in the open, the amount of light is rarely an on/off switch, which means there will be periods with varying degrees of activation. Since the bacteria should be active during overcast days, the system requires a threshold, both in the sensitivity of the light-regulated promoter, but also in the activation of the toxin-antitoxin system. Variation of light is implicitly modelled through variation in promoter strength, for instance a half probability of activation translates roughly to a half production rate in the individual bacteria. Because of this, it is important not only to find functioning configurations, but also to investigate the closely related configurations, so that the bacteria neither lay dormant at overcast days, nor make the dormancy system obsolete in moonlight.

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.

Approach

Balancing Bacterial Dormancy Requires Accurate Regulation of the System
The genes needed for inducing dormancy when the bacteria are not exposed to light, are found in the photocontrol device part, BBa_K519030. This part is composed of three genes named ho1, pcyA, and cph8, all of which are essential to ensure the cells ability to respond to red light. When the photocontrol device is exposed to light, a phosphorylation cascade activates the transcription factor OmpR, which in turn induces transcription through the OmpR-regulated promoter, BBa_R0082. This system is combined with the RelE-RelB toxin-antitoxin system in the endeavour to mediate light-dependent dormancy in bacteria. In the first considered design of the light-dependent dormancy system, the aim was to clone the photocontrol device, BBa_K519030, under control of a constitutive promoter, RelE under control of the OmpR-regulated promoter, BBa_R0082, and RelB under control of a constitutive promoter, all into one high copy BioBrick assembly plasmid pSB1C3, as seen on Figure 1.

Figure 1. All three components of the light-dependent dormancy system cloned into one high copy plasmid.

The Photocontrol Device was Placed under Control of a Constitutive Promoter
From the constitutive promoter family the weak promoter, BBa_J23114, the two medium promoters, BBa_J23106, and BBa_J23110, and the strong promoter, BBa_J23102, were tested by fluorescence microscopy to determine which one to use for expression of the photocontrol device and RelB. Overnight cultures of the submitted parts expressed in E. coli TOP10 illustrated a clear gradient of increasing red fluorescent protein (RFP) expression correlated with the strength of the promoter, as seen on Figure 2.

Figure 2. Cultures of E. coli TOP10. From left to right: WT, the weak promoter, BBa_J23114, the two medium promoters, BBa_J23110, and BBa_J23106, and the strong promoter, BBa_J23102.

On the basis of the data obtained by fluorescence microscopy, the strong constitutive promoter, BBa_J23102, was chosen to control the photocontrol device. For still inexplicable reasons the photocontrol device emerged difficult to clone with the strong constitutive promoter. Although the molecular cloning of these parts was optimised several times very few successful clonings were accomplished, and the few times correct assembly was obtained, the BioBrick was not reproducibly purifiable. By sequencing, it was deduced that a region of the plasmid containing both the promoter and the BioBrick prefix had vanished. To circumvent this inconvenient cloning two other promoters, that are constitutive in E. coli, were examined, namely the PenI-regulated, BBa_R0074, and the Mnt-regulated, BBa_R0073, promoters. By performing fluorescence microscopy on composite parts of the promoters controlling yellow fluorescent protein (YFP), BBa_I6102, and BBa_I6103, respectively, the expression levels were assessed. The obtained results revealed that the PenI-regulated promoter facilitated very strong expression of the marker gene, whereas the expression controlled by the Mnt-regulated promoter was noticeably lower. Based on these findings, the photocontrol device was placed under the control of the PenI-regulated promoter instead of the strong constitutive promoter from the constitutive promoter family. As it turned out, this cloning likewise emerged difficult. After several attempts, it was decided to focus on the other aspects of the dormancy system.

Regulation of the OmpR-dependent Promoter Required a Low Copy Vector
The first construct containing the genes required for the light-induced dormancy was designed as shown in Figure 1. As the conducted modelling clarified, the necessity for stringent regulation of the RelE and RelB expression, the properties of the OmpR-regulated promoter were studied thoroughly. To assess the functionality of the OmpR-regulated promoter in practice, a reporter system containing the OmpR-regulated promoter controlling RFP was cloned into the E. coli strain MG1655 ΔOmpR. The phenotype of the resulting cultures revealed a dysregulation of the OmpR-regulated promoter. Thorough research lead to the finding that the OmpR-dependent promoter is not controllable when cloned on a high copy vector. As the modelling revealed, and which is evident from Figure 2-Main-Page, a relatively low expression of RelE is required to induce dormancy, whereas high expression levels quickly result in overshooting. Since the OmpR-regulated promoter is an integrated part of the light sensing system, replacement is not an option. Therefore, the variability of the relE gene copy number was studied, and it was found that the OmpR-regulated promoter should be cloned into the bacterial chromosome or a low copy vector to obtain proper regulation 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.. This intriguing finding let to the aspiration to investigate the controllability of the OmpR-dependent promoter on vectors with different copy numbers compared to the chromosome, thereby improving the characterisation of the promoter for the benefit to future iGEM teams.
To incorporate DNA onto the bacterial chromosome, homologous recombination with the red λ recombinase is a suitable approach Thompson JF, de Vargas LM, Skinner SE, Landy A. Protein-protein interactions in a higher-order structure direct lambda site-specific recombination. Journal of molecular biology. 1987;195(3):481-93.. Using this technique, a short fragment of chromosomal DNA at the bacterial attachment site attB Groth AC, Calos MP. Phage integrases: biology and applications. Journal of molecular biology. 2004;335(3):667-78. can be replaced with a linear DNA fragment encoding the OmpR-dependent promoter, RelE, and an chloramphenicol resistance cassette. Using polymerase chain reaction (PCR), the linear DNA sequence was flanked by sequences, which are homologous to part of the chromosome. The linear DNA fragment was electroporated into bacteria containing the pKD46 plasmid, encoding the red λ recombinase Thompson JF, de Vargas LM, Skinner SE, Landy A. Protein-protein interactions in a higher-order structure direct lambda site-specific recombination. Journal of molecular biology. 1987;195(3):481-93., which mediated the recombination. The fundamental concept of this approach is illustrated in Figure 3.

Figure 3. The principle behind the recombination. By PCR, the two flanking sequences are assembled with the fragment containing the chloramphenicol resistance gene (camR), the OmpR-regulated promoter, and the relE gene. The flanking sequences are homologous to part of the chromosome around the bacterial attachment site (attB), enabling the homologous recombination.

Using this approach, it was attempted to assemble the OmpR-regulated promoter controlling RelE with the flanking regions by PCR. The assembly of the fragment derived from the plasmid with either one of the flanking fragments was achieved. However, the assembly of the complete chromosomal insertion fragment containing all three segments emerged problematic. Several attempts with numerous process optimisations were performed, but unfortunately without success. To circumvent this, another attachment site was chosen. The assembly of the complete fragment by PCR was achieved and chromosomal insertion by electroporation was attempted several times, but in vain. In consideration of the fact that RelE is a bacterial toxin, the cloning of this gene was inconvenient. The model indicated, that overexpression of the antitoxin RelB would bypass this difficulty. As the homologous recombination of this fragment emerged such a challenging task, it was decided to focus on the conduct of the OmpR-regulated promoter on different plasmids, as this was evidently the more convenient approach of the general implementation of the OmpR-regulated promoter.

An Inducible Promoter was Chosen to Regulate the Gene Expression of the Antitoxin RelB
Originally, it was thought to place the antitoxin RelB under a constitutive promoter of appropriate strength. However, as the modelling revealed, strict regulation of RelB is essential to counteract the toxic effect of RelE and enable a functional dormancy system. Thus, it was deduced that it would be more suitable to utilise an inducible promoter for this purpose and it was decided to put RelB under control of the LacI-regulated, lambda pL hybrid promoter, BBa_R0011. The William and Mary team found that the LacI-regulated, lambda pL hybrid promoter had a low level of noise, a measure of the variability in gene expression between cells in the population, when cloned into a low copy vector. Therefore, this regulated promoter was chosen.
To mimic the expression of RelB, a reporter system composed of the LacI-regulated, lambda pL hybrid promoter and GFP was assembled. Hereby, the appropriate concentration of IPTG required to induce the promoter on a low copy vector, was identified. During the cloning, it was discovered that the site between the LacI-regulated, lambda hybrid promoter and this particular construct under the tested conditions had formed a hotspot for transposons. In the light of this finding, another inducible promoter was chosen. The HKUST-Rice iGEM 2015 team demonstrated that induction of the AraC-regulated promoter, pBAD, caused a gradual increase in gene expression when cloned into the low to medium copy plasmid, pSB3K3, in contrast to an all-or-none behavior when cloned into the high copy vector, pSB1K3. Taking these results into consideration, the pBAD promoter was used in combination with the pSB3K3 vector to regulate the expression of the antitoxin RelB. Again, a reporter system was used to mimic the expression of RelB, as the part, BBa_I6058, containing YFP controlled by pBAD, was assessed on different vectors.

The Final Approach for the Three Components Comprised Three Different Vectors
Based on the modelling the system approaches reviewed in the preceding part, the final design, which is illustrated on Figure 4, was established. Ultimately, the dormancy system was composed of the photocontrol device controlled by the PenI-regulated promoter on a high copy vector, RelB controlled by pBAD, BBa_K2449031, on a low copy vector and RelE controlled by the OmpR-regulated promoter on either a low copy vector or the chromosome.

Figure 4. 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 plasmid.

Team:SDU-Denmark/Model

Project Design

Dormancy System

Light-Dependent Dormancy System

Gillespie Algorithm

Stochastic Modelling of the RelE-RelB Dormancy System

Modelling Results of the RelE-RelB System

Light Sensing System