Future Directions

Making a better switch

Push-button Switch

It is possible to construct the switch such that brief exposure with an inducer produces a burst of recombinase expression, which in turn act upon the switching sequence and forming a sustained switch. An example of such a mechanism would utilize constitutively expressed repressors to suppress recombinase expression until the inducer temporarily inhibits repression.

Positive Feedback Loop

Leaky expression of each recombinase when under an inducible promoter may cause a significant fraction of the cell population to have a switch flipped in the wrong direction if the recombinase leakage is above the concentration threshold necessary to flip the switch. From our model, we concluded that leaky expression will eventually reduce the system to one of mixed states. In order to remedy this problem, we propose a positive feedback mechanism. In addition to the desired genes to be expressed on either side of the switch, one could add the recombinase that will flip the switch back to its current state in the event that undesirable basal level expression of the opposite recombinase flips the switch. This will create a threshold level of recombinase activity that will need to be overcome by the opposite recombinase in order to completely flip the switch. Extra consideration would have to be given to the transcription and translation rates involved in a circuit like this. The threshold level of recombinase activity must not be so high that the opposite recombinase is unable to overcome this activity and flip the switch. Too high of a threshold may be a burden on the cell as well. On the other hand, the threshold level must not be so low that the basal level expression of the opposite recombinase is able to flip the switch to the wrong state.

Antisense RNA (asRNA)

Alternatively, antisense RNA (asRNA) can be used to generate a threshold level of leaky recombinase gene transcription such that a higher level of recombinase expression is required before significant switching can occur. Expression of the competing recombinase can be reduced if downstream of the switching promoter is a sequence that produces low levels of mRNA complementary to the RBS of that competing recombinase, effectively acting as an asRNA. The competing recombinase must transcribe at higher levels before it can overcome the effects of asRNA. When the competing recombinase mRNA level passes the threshold level posed by the asRNA, recombinase proteins will be produced such that a switch occurs, at which time the asRNA of the now competing recombinase will be expressed.

Fine tuning promoter and RBS strengths

Combination of promoter and RBS driving recombinase expression could also be fine-tuned such that each recombinase produces sufficient flipping when induced with minimal leakiness when not induced.

Degradation Tags

One could imagine wanting to flip the switch in rapid succession. If the recombinase from a previous flipping is still present in the cell, this may delay the amount of time it takes to flip the switch to the desired state. In order to increase the ‘responsiveness’ of the circuit, degradation tags can be attached to the recombinases that will tell the cell to degrade them faster. These will prevent unwanted recombinases from sitting around in the cell too long. One must consider how this will affect flipping of the switch though. If the turnover rate of the recombinases are too high, there may not be enough time for them to flip the switch.

Recombination sites around the inducible promoters

Adding LRP and IHF sites

DNA-bending proteins such as leucine-responsive regulatory proteins (Lrp) and integration host factor (IHF) protein natively interact with fimS in fimbriae producing E. coli. These proteins could bind close to and assist in creating a loop encapsulating the switching sequence, bringing IRR and IRL sites closer together. This additional component may increase the rate at which the tyrosine recombinases form a Holliday junction and flip the switching sequence.
For more information on tyrosine recombinases forming Holliday junctions, see: Holden, N et al. Comparative analysis of FimB and FimE recombinase activity. Microbiology. 2007 Dec;153(Pt 12):4138-49. doi:10.1099/mic.0.2007/010363-0

Chromatin remodeling to control recombinases (Eukaryotes)

Future Impacts

The ability to accurately control the onset and termination of expression for proteins of interest enables further automation on cell/virus-based protein synthesis systems, opening opportunities for novel or better optimized high or low-throughput applications. I imagine a simultaneous induced switch flipping in a 96-well microplate, which is particularly beneficial for time sensitive assays and also serves to reduce the number of samples required for meaningful statistical analysis.

Making More Complex Circuits

N recombinase pairs gives 2^N states