Team:ESBS-Strasbourg/Project/Application

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4. Light controllable protease

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As previously described, our degradation system consists of an engineered protease which can be activated by light impulses. This allows a tight control over the catalytic activity core enabling the modulation of protein function in a general fashion with the combined characteristics of specificity, high temporal precision and rapid reversibility. Illumination with far-red light could lead to disrupt the interaction between PhyB and PIF3 and to thus switch off degradation.
The system is easily adaptable to new targets proteins, the target-labeling only requires the fusion to the specific degradation tag and PIF. This offers a very cheap easy and applicable method for protein analysis.

One of the major advantages is the "non invasive" induction of the protein degradation. Chemical genetics enable perturbations through the introduction of cell membrane-permeable small molecules, allowing the conditional regulation of activity through non-covalent and reversible interactions which is convenient for studies at the cellular level. The use of photolabile ‘‘caged’’ chemical compounds allows to affect subcellular targets in a second-timescale. Some chemical photoswitches such as azobenzene even offer reversible photo-control when attached to macromolecules [2]. However, the requirement to introduce exogenous, chemically modified materials into cells limits the use of these methods in biological applications.
Instead of the induction by chemical agents, the induction of our system is achieved by light impulses. Chemical agents can interfere with host cell metabolism thereby changing their behavior and impact on complex pathways which may create the impossibility of obtaining neutral results. The induction by light enables the studies of target proteins in a natural unaffected environment.

Another alternative in protein function studies is the use of gene-knockout techniques. These approaches can provide information about incompletely known gene functions, for instance the role of the corresponding protein in interactions with other proteins. But they do not provide any possibility to study kinetic characteristics or the dynamic of protein interactions.
Our system provides a very effective alternative to this approach. Due to the possibility to regulate protein degradation by light-guided on/off switching of the protease activity, it is a tool to control the level of target protein concentration. The common gene knock out methods do not provide any insight to the impact of varying protein concentration. Moreover, suppressions of a protein by recombination or CreLox methods are more difficult to set up, as the suppression is irreversible it can be lethal for the cells.

This new system allows through its high turnover rate for proteins [1] a complete degradation of the protein, simulating a gene knockdown. After light induction with 660nm the system should rest in its active state until a light impulse of 730nm changes its back on its inactive state. So a permanent on switch simulates a gene knockdown as every protein is immediately degraded and a permanent off switch favors the native gene expression.
br> With alternating light impulses it should be also possible to adjust certain protein levels by switching the system on and off. This allows the control of complex protein dynamics in vivo as all protein levels can be adjusted to simulate the desired condition.

Such a system is useful in any domain of research. Both medical researches as fundamental cell biology require a deep understanding of protein function and their role in interactions with other proteins as in signal cascades and metabolic pathways. The possibility to control protein dynamics in a general manner offers a great approach for medical treatments.

An example of this tightly controlled system can be seen in figure 1.

Figure 1 An example of how protein levels can be adjusted by alternating light impulses.
In the beginning, the protein levels are at native concentration. After a light impulse the degradation system
is on and will degrade the protein very fast and efficient. These first two steps are like a gene knock out
with an on and off switch. After this an alternation of light impulses turn the system on and off in certain
time periods. So the protease is turned between active and inactive. This allows the fine tuned adjustment
of protein concentration in the cells.

Flip Flop

The system further allows the control of transcriptional regulation. Another application of this system is the creating of a flip flop mechanism which can be induced by light. This can allow the expression of two different genes sequentially. In the beginning just the gene in gene cassette one is expressed. In the example this is the GPF protein. After a light induction the gene expression is switched to gene cassette two, which is RFP in this example. Figure 2 gives a more detailed description of this mechanism. This allows the tight control of two genes in one host organism. The tight control and sequentially nature of this flip flop mechanism allows a light-controlled multistep synthesis which a huge potential for industrial application in multi -step synthesizes.
Moreover several enzymatic steps can be conducted sequentially in one single organism, so even complex biomolecules can be produced in a single bioreactor. This is an enormous gain of time and money.


Figure 2 The flip flop mechanism. This mechanism shows how to change from the expression of a
gene in the first cassette to a gene in the second cassette. P is the promoter, CR is a cross repressor,
the symbol besides the cross repressor symbolize that this protein is tagged with the DAS degradation sequence,
CA is a cross activator and C is the gene cassette. At start condition P1 expresses all the proteins of gene
cassette one (C1). The cross repressor for promoter P2 (CR2) represses P2 stronger than the cross activator
for P2 (CA2) activates it. This results in an expression of the GFP protein. After light induction with 660nm, the ClpXP
protease will degrade the tagged CR2. After the degradation of the repressor, the cross activator will activate
the promoter P2 which will lead to an complete expression of gene cassette two (C2). The CR1 of the C2 will now repress
P1 which will terminate the expression of gene cassette one. So a switch from C1 to C2 is achieved. An light impulse
of 730nm will switch of the ClpXP protease. With another light impulse of 660nm the ClpXP system will be turned on
and a switch from C2 to C1 will occur. A detailed analysis of this mechanism can be seen in the modeling part.

Genetic Oscillator

The idea of the flip flop mechanism can be extended to a genetic oscillator with three, four or even more sequential steps. Figure 3 shows an example of a three step oscillator. This oscillator is tightly controlled by light and allows the sequentially expression of three different genes. Such an implementation would present a genetically encoded device to store multiple bits of information within a living cell.

The light-dependent protease with its specific degradation tags is a versatile approach for transcriptional regulation and protein analysis. It gives the synthetic biology community a basic device with a broad range of applications in fundamental research. The only limit is the imagination, the motivation and the skills.


Figure 3 The three step oscillator. The principle is the same as with the flip flop mechanism. In the beginning
gene cassette C1 with GFP is expressed and CR2 and CR3 represses P2 and P3. After a light impulse of 660nm,
CR2 and CR3 are degraded and CA2 can activate P2. The ClpXP system will be switch off by a light impulse with 730nm.
Due to the absence of CR2 and CR3 gene cassette C2 and C3 will be no longer repressed. But as just an CA for
the P2 was expressed from C1, C2 will be far stronger expressed than C3. So the CR3 on the C2 will terminate gene
expression of P3 and thus will terminate the whole expression of C3. CR1 will also repress the expression of P1 and thus the
whole expression of C1. After another light impulse of 660nm, the switch from gene cassette two (C1) to gene cassette
three (C3) will occur with the same mechanism as from C1 to C2.

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