Designs overview

Introduction

Our goal for this summer is characterizing the exchange of proteins through nanotubes as best as we can. In order to do that, we want to construct several design. In this page, we are going to explain you the principle of the different designs we have builded.

In the original paper, the author observe directly the molecules that passes through the nanotubes. The idea behind our approach is to use synthetic biology methods to go farther on the sensitivity and the resolution of these experiments. Relying on signal amplification methods and natural and artificial bistable switches to detect at the molecule resolution when a few molecules have diffused from one emittor cell to the receiver.

Main questions behinds

The nanotube discorevy is very recent. Though, lots of question remains unsolved, on the processes of the formation of the tubes, the efficiency of the communication, the extent of the process. The existence itself remains contested by some scientists.

We aim to add additionnal proof of their existence and increase the number of the nanotubes, and characterize better the extent of the phenomenon. Here is the list of the question we are asking ourself, and that our designs aimed to answers:

How the tubes are forming?
What is the speed of the process? (passive or active transport)
Can we pass all kind of molecules?
Is the communication happens only in between B. subtilis, or can it happens also interspecies or even between E. coli strains?
If a communication is established with a Gram positive bacteria, is the communication happening through the periplasm of the cytoplasm?

Specification of our designs

We started designing the project with these the previously stated questions in questions in mind. Using several molecules of different sizes, from a T7 polymerase to a tRNA amber supressor, we aim to test the capability and size limit for the molecules that can pass through the tubes. But, if the diffusion is passive, the speed of diffusion should be proportional to the diffusion coefficient D, that is to say inversely proportional to the radius of the object. Can we design a system fast-responding enough that would allow us to measure this speed of the transfert?

We decided that our constructs should follow the global idea summed up in the following picture:

Fig1: General plan for the experiment design to characterize the nanotubes

The problem is that each design needs a different couple of signal emitter/receptor. However, even with good models, the response time cannot be predicted easily because little is known about the transfer through the nanotubes. We tried to limit the impact of this issue by the following measures:

Use each time the same actuator and the same monitor to have comparable response times in the different systems.
The relevant measure is not the response time itself, but the increase of response time when the two constructs are in the same cell or in different cells linked by a nanotube

The idea is if we can measure this increase of time for different sized, that is to say of different D coefficien, of the molecule we should get a relation in lambda/D, the diffusion is an active process. Else, the process is active. See the modeling pages for more details about this assuption.

In B.subtilis, nanotubes are reported to be formed by the cytoplasm. We had to find a molecule that can be expressed in the first cell that can trigger a genetic circuit in the second cell. The image below illustrates the idea:

Fig2: Schematic of the supposed connection between the cells via the nanotubes

Building fast new genetic devices

Using these specification, we designed several sensitive genetic circuit that can be trigerred by molecules of various size. The molecules chosen cover 2 orders of magnitude of Radius. Here, they are classified from the biggest to the smallest.

		Type of switch	B. subilis E. coli compatibility
	T7 polymerase diffusion: The T7 RNA polymerase is the RNA polymerase of the T7 phage. This is a big molecule, that recognizes a very specific promoter orthogonal to B.subtilis.	Irreversible amplification	Yes
	Amber suppressor tRNA diffusion: The principle of this design is to produce in one cell an amber supressor tRNA that will diffuse through the nanotubes. The receptor cell holds the gene for T7 with amber stop codons that cannot be translated into a functional protein as long as the tRNA amber suppressor is not present in the cell. Once expressed, the functional amber T7 RNA polymerase will trigger the T7 amplification system.	Irreversible amplification	Yes
	KinA diffusion: The Sin operon system controls the sporulation switch of B. subtilis. Making it diffuse from a non sporulating strain to a receiver cell makes the receiver cell to sporulate.	Switch	Mono- directional
Lambda switch	CI diffusion: The CI is a regulation protein from the lambda phage switch. Make it diffuse would change the state of the toogle switch from the push-on push-off system, from the PKU 2007.	Switch	Mono- directional
	The YFP-TetR/TetO array experiment: This experiment is an improvement of the previous one. To observe significant fluorescence in the neighboring cell, lots of molecules have to pass through the tubes. Using the affinity of the TetR for the TetO array, we want to concentrate at one point the YFP molecules to better observe this diffusion.	-	Yes
	ComS diffusion: ComS is an inhibitor of the MecA protease in B.subtilis. It plays a key role in the triggering of the competence and sporulation mechanisms. The idea is to trigger the switch of the MeKS system of the receptor cell by diffusing ComS through the nanotubes. A major precaution is to prevent the first cell from sporulating.	Switch	Mono- directional

Others designs we had no time to build

We neither had time nor the manpower to try all the ideas we had. Some designs too complicated to be feasible. We also wanted to investigate at length the Subtilis/Coli connexions but had no time to do it properly. We nonetheless present all our ideas in this section.

Abandonned design for B.subtilis

Xis protein diffusion: Xis is a small partner of an excisase. The latter will excise a stop codon on the DNA strand that is preventing the expression of the GFP. We had no time to build this design, but the idea is summed up on the linked page.

Designs for B.subtilis-E.coli communication

The article demonstrates that nanotubes established inside the B.subtilis family can also be formed between B.subtilis and E.coli. These two types of bacteria are really different since one is Gram+ and the other is Gram-. Their membrane is really different since there is no periplasm in B.subtilis. The existence of inter-species nanotubes did really spike our interest and we also created designs to test this aspect of the nanotube communication. We had few time to test these ideas but we present here the designs we thought of. For all the designs which are simply the ones for B.subtilis applied to a Subtilis/Coli connexion, we plan to experiment them in the future since they require very little additional work.

B.subtilis is Gram- so the nanotubes seem to create a link from the cytoplasm of the first cell to the cytoplasm of the second cell. In the case of the Gram+ bacteria, like E.coli, we wonder which membrane forms the nanotubes . Do they create a link with the periplasm, or with the cytoplasm? To test the two hypotheses, we tried to create a design for each of the two possibilities. If one work and the other does not, the answer to this question will be known.

If nanotube communication can be established between E.coli and B.subtilis, it is not known whether the communication happens through the cytoplasm or the periplasm. To test these two hypotheses, two designs were made:

If communication happens with the cytoplasm

The image below sums up the kind of connection that can be established if nanotubes connect the two cytoplasms. Hence, some of the designs we have shown above are still available for B.subtilis to E.coli.

Fig3: Schematics of the communication happening directly to the cytoplasm

Here are the designs created in this case.

T7 polymerase diffusion: The T7 RNA polymerase is the RNA polymerase of the T7 phage. This is a pretty big molecule, that recognizes a very specific promoter orthogonal to B.subtilis and E.coli.
KinA diffusion: The idea is to make a complex partner to diffuse from one cell to the other in order to trigger the sporulation of the second cell, even in exponential phase.
ComS diffusion: ComS is an inhibitor of the MecA protease in B.subtilis. It plays a key role in the triggering of the competence and sporulation mechanisms. The idea is to trigger the switch of the MeKS system of the receptor cell by diffusing ComS through the nanotubes. A major precaution is to prevent the first cell from sporulating.
Amber suppressor tRNA diffusion: The principle of this design is to produce in one cell an amber suppressor tRNA that will diffuse through the nanotubes. The receptor cell holds the gene for a T7 with amber stop codons that cannot be translated into a functional protein as long as the tRNA amber suppressor is not present in the cell. Once expressed, the functional amber T7 RNA polymerase will trigger the T7 amplification system.

If communication happens with the periplasm

In case the communication happens with the periplasm, we have to think about molecules that can be then transported into the cytoplasm.

Fig4: Schematic of the communication happening through the periplasm.

We had to design more sophisticated approaches. The ideas are the following:

MBP diffusion: we need a CRP+, MBP- E.coli mutant. We produce the MBP protein in B.subtilis and make it diffuse through the nanotubes. As long as the MBP has not reached the periplasm of E.coli, the cell cannot digest the maltose in the medium. The indirect induction of MalR by MBP triggers the expression of the GFP reporter.
OmpR diffusion: we need a OmpR- Receptor* E.coli mutant. We produce the OmpR protein in B.subtilis. As long as the OmpR has not diffused from B.subtilis, the signaling cascade cannot be activated. With the rescue by B.subtilis of the OmpR protein, the expression of the reporter gene is activated.

From electronic microscopy images, the author shows that the communication through the nanotubes can also occurs inter bacteria species. We also designed our experiments so that we can support the existence these interspecies communication with optical microscopy experiments, by placing our emitters and receivers either into B. subtilis and E. coli.

Can we conclude on these designs?

If one of these designs works, it will be proved that the inter-species nanotube communication exists and this will eventually show location of the connection with the membrane. There was limited time to test these designs but we propose them for future iGEM teams to investigate.