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Module I - HOTFM

We call this module ’Highly Optimized tunable Frequency Modulator (HOTFM)’. The idea is to modify the Danino Oscillator so that we can tune the frequency of os- cillations. Further, we wish to use optogenetics to control this tuning. Oscillators can be used as clock references, and thus, controlling the frequency of oscillation would offer certain advantages. For instance, increasing the frequency might allow faster execution of a process and vice versa. In this regard, one possible medicalapplication could be time-controlled drug delivery. If a drug is known to function optimally if delivered at distinct regular intervals, an oscillator handling the de- livery of the drug would be an attractive option. Further, if under circumstances quie stark from the ordinary, the rate delivery needs to be increased (decreased), our approach could be a viable option.

We require two additions to an oscillator to realize HOTFM. A mechanism to modulate the frequency of oscillation and an optogenetic module to act as a switch for this mechanism.

Frequency Modulation

The Danino oscillator has a latency period where both the activator (LuxI) and the repressor (AiiA) grow in concentration. One possible way to reduce the time period of oscillations would be to reduce this latency period. For the Danino os- cillator, it is known that the time period and amplitude have a linear relationship [4]; thus, we could, in principle, reduce the amplitude to reduce the latency pe- riod and increase the frequency of oscillation. We propose to do this by adding negative autoregulation on AiiA. This transforms the amplified negative feedback oscillator into a smolen oscillator [8]. There are additional benefits of adding negative autoregulaation on AiiA; this makes the oscillator robust to parameter variability [8]. If there is a sudden variation in AiiA’s concentration the negative auto-regulation would automatically bring the concentration back down. Thus, we expect HOTFM to have lesser peak to peak variability compared to the original Danino oscillator. In-silico simulations confirm our intuition about HOTFM (refer to the modelling section for a detailed analysis).

Optogenetic Module

In order to have a tunable frequency modulator, we need to have a switch that controls the negative auto-regulaation (self-repression) on AiiA. To achieve this end, we use optogentics as a flexible and easy mechanism. The Self-repression pathway is put under the control of lambda repressor; and this lambda repressor controlled by the Ccas-Ccar system, which is an optogenetic module. So, whenever the oscillator is required to change it’s frequency of operation, we can, in principle, shine light on the cells and the self-repression pathway (mediated by the lambda repressor) would be activated.

iDanino -Synchronized oscillator and toggle switch Biological implementation

Synchronized Oscillator

Our synchronized oscillator is highly inspired by the work of Tal Danino [11]. Broadly, it is based on the quorum sensing machinery of Vibrio fisheri and Bacillus Thuringiensis. LuxI(from V.fisheri) under lux promoter , aiiA(from B.Thurigienesis),and mRFP1(from Discosoma sp.) genes under the control of Plux-lambda hybrid promoter (Part:BBa_K415032). LuxI, aiiA, mRFP1 with degradation tags were used, as it was clear from initial modelling that accumulation of these would disrupt the oscillations.

The LuxI protein enzymatically produces a small molecule acyl-homoserine lactone (AHL) which can easily diffuse through membrane and facilitates the cell to cell communication. AHL works in conjugation with the protein LuxR(from V.fisheri) by forming a complex with it, which after binding can bind to the operator site in the LuxI promoter and activate it, increasing the rate of transcription of the gene placed downstream to this. In our system, AHL intracellularly binds to constitutively expressed LuxR to form the LuxR-AHL complex which is used as a transcriptional activator of Plux-lambda hybrid promoter [12]. AiiA negatively regulates the transcription of the hybrid promoter by degrading AHL [13,14] molecules.

Oscillations of this system were visualized using microscopy and microfluidics. After an initial transient period, stable synchronized oscillations could be seen in our bacterial culture. The dynamics of the oscillations can be interpreted in the following manner:

Since AHL is a small molecule which can diffuse across the cells, it communicates with the other cells in the vicinity of the cell that it diffused out of. aiiA degrades the AHL internally[15], and consequentially, a small colony of cells can’t activate oscillations as whatever AHL diffuses into the cell is quickly degraded by the aiiA protein. However, as the concentration of AHL increases, it activates the luxI promoter, causing a sudden transcriptional burst of genes- aiiA, LuxI, mRFP1- under luxI promoter. Now as aiiA accumulates, it starts degrading AHL and after a certain time, the concentration of AHL goes below a certain threshold, such that it can no longer activate transcription. The luxI then returns to its inactivated state. Following this, there is another decrease in concentration of aiiA (due to absence of the activator), which leads to loss of repression of the luxI promoter and hence causes an increase in the production of AHL, due to which there is another burst of LuxI, and in this manner, the system oscillates.

Toggle Switch
For the construction of the programmable toggle switch and in order to have more control over the system, another circuit was implemented in the cell. In this circuit, a heat sensitive Lambda repressor CI (BBa_K098995) was constitutively expressed under the strong promoter BBa_J23101. [16] At 37 degree Celsius, CI lambda repressor- undergoes complete thermal denaturation and therefore cannot bind to the operator site of the lambda repressible promoter, or a hybrid lambda promoter. Therefore, at 37 degree Celsius, there is no transcriptional repression due to CI.

The choice for the circuit was done as follows. Upon reading literature, having decided to design programmable oscillator circuits, we inferred that due to the mode of action of LuxI, it would be difficult to have the system produce anything at the steady state if we were to stop the production of LuxI to generate the toggle (as LuxI produces AHLs, which drive the whole circuit). We therefore decided to repress the production of the aiiA protein.

The methods considered for the repression of aiiA were :
To degrade aiiA at the post translational stage – This could be achieved using protein-protein interaction circuits, for example the circuit developed by Ron Weiss et al[23]. However, due to their complex mode of action and difficulty to Engineer reversibility, we decided against using it.

To stop production of aiiA at the transcriptional stage – This was a good idea, as the interaction was simple, using any of the large number of repressive transcriptional factors, which are usually easy to reverse. We therefore decided to use this method for constructing the toggle switch.

The lambda repressor (CI) was chosen due to its easy availability along with an LVA degradation tag, and simple reversibility on increasing temperature. Hence, we decided to go for this system.

The working of the system is as follows. At 37 degrees Celcius, the CI repressor, as mentioned above, is completely denatured, and therefore the oscillations continue normally, similar in order of time and space as the Danino oscillator. However, once the culture is brought down to 30 degrees Celcius, the CI is no longer denatured, and becomes completely active. Thus, all the aiiA is repressed, and the system constantly amplifies the production of LuxI and toggles the LuxI into the ON position, and the aiiA to the OFF position. Connecting a reporter across either one gives an ON/OFF position at 30 degrees, as needed.

Strain Used:
In all our experiments Dh5-Alpha from NEB was used.

Plasmid Construction
To implement the system in E.coli, we designed the plasmids as shown in the figure. LuxI, LuxR and mRFP1 were put on psb1C3 which has pmb1 origin of replication and is high copy number. Repressor aiiA and lambda gene were put on psb3T5 backbone as it has p15A ori, which is low copy number plasmid. Only through modelling it was figured out that aiiA repressor should be on low copy number plasmid and as lambda repressor is repressing so aiiA so it too was put on same plasmid. All the parts were procured from registry.

part retrieved from registry was faulty. We have successfully characterized by verifying its plasmid, single digestion and double digestion on gel.
Part name
LuxI (+LVA) - Registry part was used
LuxR(+LVA) - Ordered From IDT
aiiA(+LVA) - Registry part was used
Plux-lambda - Registry part submitted by Tabor was used.
C1-lambda(+LVA) - Registry part was used
mRFP1(+DAS) - Registry part was used
All the clones were verified on agarose Gel. The images of clones are there in results. Clones were also sequenced for verification.
Future implementation - HOT-FM
As mentioned earlier we wish to regulate self-repression using light.

Optogenetically controlled Self-Repression

In a line, idea was to control the self-repression of component B using light. For this purpose, Lambda repressor was expressed under the control of light sensitive CcaS-CcaR(from Synechocystis PCC 6803) system. It is a two component green light sensitive system which activates the promoter pCPCG2 in green light and gets repressed in red light [17]. We used temperature sensitive Lambda-851 repressor so as to have an added control of temperature.

It was positively regulating the expression of lambda repressor by activating its transcription. CcaS-CcaR system itself is regulated by Plux-Lambda(………..) so CcaS- CcaR gets activated by LuxR-AHL complex but it will bind to cpcG2 promoter only when green light is shone i.e Lambda repressor will be expressed only when system is illuminated with green light.

We have extensively modelled the circuit and designed biological circuit. It consists of three plasmids two of them are ready and has been submitted to iGEM registry. Remaining plasmid which contains optogenetic part has been ordered from GENESCRIPT but it we haven’t received it so far.

Team:IIT Delhi/Modules