When studying the impact or function of particular genes, sometimes just being able to have them be expressed isn’t good enough. An always on state can be just as frustrating and produce just as useless data as an off state would. This is especially true for signaling and transportation genes, where the entire point of studying them has to do with being able to view the precise moment of action that the transcribed genes result in through their protein activity.
Therefore, direct genetic manipulation isn’t the way to go for this purpose, as there’s no way to only partially express a gene when you want directly that way. Only an epigenetic system would have the possibility of achieving that result. In comes an option that was named Method of the Year in 2010 for it broad applicability in multiple fields of study, essentially anything that worked with cells and genetics. That method is known as optogenetics, the alteration of genes by using waves of light.
Optogenetics: The Power of Light
Gene expression through exposure to light is not anything new, in fact, it could be said to be one of the oldest mechanisms in the biological world stemming back to early plants and cyanobacteria. But its use in animal cells has always been more limited. We lack the chromophore receptors needed in most cases to have such a response, as only red and infrared light is truly capable of penetrating human skin and reaching the cells in interior tissues. These receptors are largely only found in the aforementioned plants.
The chromophores needed for this purpose are based on phytobilin, light-capturing receptors from algae species. The specific components required for gene control have, in past studies, been the receptors phycocyanobilin (PCB) and phytochromobilin (PΦB), which are produced by several enzymes in combination. The most common enzyme used in these optogenetic systems has been ferredoxin (Fd), an electron transporter that is able to supply energy from the captured light to the other enzymes so they fulfill their purpose in altering genetic output.
These prior studies have used transgenes of the enzymes from algae and transferred them into animal cells for studying certain genes. But the intermittent expression has been hard to control and does not always function as desired or only has expression last for minutes, making researchers scramble for measurements in the short time period.
An Unconsidered Variable
Scientists at the University of California have presented an alternative process that maximizes control while minimizing the inputs needed. What they figured out is that all the prior research had been ignoring the impact that ferredoxin has on chromophore production. It had been presumed, seemingly without evidence, that the limiting factor in this production was heme, as a byproduct of heme is needed in the chromophore production process. So attempts had been made to localize where the enzymes were placed to be in the same place as where heme is made in order to enhance creation of PCB. However, these previous researchers were just not getting the increased gains they were expecting.
The UC San Diego scientists looked into how most cells have endogenous ferredoxin in them, but these are not all identical. Fd seems to be selective on what enzymes it will interact with based on the species it is found in. So animal Fd is practically useless if you are using plant or algae-based phytobilin enzymes. This means that, rather than heme and its byproducts being the primary limiting factor in PCB production, it is actually the Fd and the related FNR reduction system that are the roadblock for increasing chromophore response for gene activation.
A New Genetic Switch
To illustrate the accuracy of this new information, they tested a PhyB optogenetic system in animal cells, where the sensitivity the PhyB receptor has to light is based directly on its concentration in the cell, as would be expected. In order to be able to use PhyB in such a system, a high amount of it needs to be made through the chromophore production mechanisms, far higher than other studies had been able to consistently achieve.
But the new system, made with Fd limitation in mind, led the UC San Diego scientists to easy accomplishment in this endeavor. They were even able to implement a two-hybrid gene switch using PhyB and PIF3, a far more complex method of controlling the light usage and how the desired genes are expressed. The necessary enzymes were transgenically added to human kidney cell cultures using plasmids via the process of lipofection to get them into the cells. Then, after further culturing, their light response was tested using red LEDs.
The real meaning behind this two level genetic switch system is how it is highly sensitive to light changes and requires low intensities of red light, it can keep genes activated for time period lasting hours if necessary, its switch makeup means that the activation can be reversed by using a particular infrared trigger, and it is able to focus on the wavelengths that best penetrate through animal tissues. All of these make this switch adaptable and controllable, far beyond what other optogenetic methods have been able to make.
Genetic Controls For Genetic Production
Finally, by increasing enzymatic output, this system doesn’t need additional supplementation with necessary chemical inputs for its activity, unlike earlier systems. The researchers believe that this proper matchup of the species-specific Fd trangene producers with the chromophore enzymes allows for the increased activation of genes related to biomolecule metabolism and transport, along with possibly producing these molecules in other cells outside their own.
If done properly, these new biomolecules have the potential for application in agriculture, pharmaceuticals, and plenty of other things within synthetic biology itself as well.
Photo CCs: Chromatophores from Wikimedia Commons