Synthetic biology really needs the equivalent of a personal computer. Sure, the costs of DNA synthesis and sequencing are dropping quickly, and many of the methods of molecular biology are becoming increasingly accessible with even a crude laboratory setup. However, synthetic biology is still big, slow, and expensive.

I’m hopeful that we can invent a personal computer equivalent for synthetic biology, and here I’ll describe what I think that might look like. Some of the parts are relatively old tech, while the creation of others would be monumental feats of protein engineering. But, I still think it would be possible.

Minimum Requirements

For a synthetic biology machine (SBM) to work, it would need at minimum water, oxygen, phosphorous, a carbon source (e.g. carbon dioxide or starch), a nitrogen source (e.g. nitrogen or urea), and some trace minerals. From these few parts, a single hardy strain of bacteria can create everything else it needs, including membranes, protein, DNA, and RNA. For the SBM to be useful to us, we need a way to send and receive information from the resident organisms in the device. The SBM would need to be sterile except for the resident organism, and maintained at a constant temperature. Ideally, the SBM would be a nearly closed system requiring only electricity–much like a tiny space ship for single celled organisms.

Information Exchange Interface

To exchange information between the user and the resident organism, it seems the easiest route would be via light (22359157). Light can be projected in a spatially controlled way, at various wavelengths, durations, and intensities–all of which can encode information for the resident organism.

A communication setup could simply use a digital projector as a sender and a camera as a receiver. The projector could display patterns onto a two dimensional field that houses the resident organism. The camera could then collect light that transmits through the organism layer. A schematic of this setup is shown in Figure 1. A more complex variant could use a 2-photon microscopy variant to query a three dimensional volume.

Input Patterns

The SBM user can communicate with and control the resident organism by changing the patterns of projected light. In many ways, this input approach is similar to photolithography, but with a wider vocabulary.

Some examples of useful data shuffling commands might be:

Become stationary

Become motile

Migrate toward the light

Maintenance commands might look like:

Die

Grow/divide

Conjugate (sex)

Degrade biofilm

One could also encode commands for synthesizing or sequencing DNA:

Form DNA synthesis/sequencing complex

Report next base in sequence

Add “A”

Add “T”

Add “G”

Add “C”

Break DNA synthesis/sequencing complex

Each of these commands would be interpreted by an engineered light-sensitive protein. I’ll describe what these might look like in more detail later.

Output Patterns

The resident organisms in the SBM can then communicate with the user by either emitting light directly, blocking certain wavelengths, or fluorescing.

Some examples of useful maintenance outputs would be:

Cell stress: “I’m about to die”

Hungry/tired : “Let me rest and regenerate”

The cell could also send DNA sequencing data out:

Read “A”

Read “T”

Read “G”

Read “C”

Beyond these basic functional responses, there are nearly infinite customized outputs that are possible if one can synthesize and sequence DNA in vivo. For example:

Report concentration of protein X

Report activity of promoter Y

Report concentration of substrate Z

Report FRET signal between proteins A and B

Each of these reporters would be engineered to do a specific thing. The sequences for each of these reporters could be collected and shared just like a software program. The program could then be loaded into the SBM by inducing the resident organism to synthesize the appropriate parts, then run them.

Minimum Set of New Proteins

Light Directed DNA Synthesis

The key part in the SBM is the ability to do in vivo light directed DNA synthesis. Once the user can direct DNA synthesis, then it is possible to bootstrap the resident organism into building all of the remaining parts.

Admittedly, engineering a light directed DNA synthesis protein complex is a monumentally difficult task, but it may not be as hard as it looks for the following three reasons. First, we already have good examples of DNA synthesis machinery in the form of DNA polymerase. DNA polymerase is responsible for copying and maintaining the genome. The sequence, structure, and function of DNA polymerase is well studied for a wide variety of organisms.

Second, the field of optogenetics has identified numerous methods for engineering light responsive protein structures. Optogenitics has primarily focused on engineering variants of the light sensitive opsin receptor–a member of which we use for vision. Beyond opsin, many other light sensing families of proteins have been identified including proteorhodopsin, cryptochrome, phytochrome, phototropin, channelrhodopsin, halorhodposin, bacteriorhodopsin, and the flavoproteins.

Third, light responsive protein design could be carried out by directed evolution. Directed evolution is a laboratory technique in which one creates large numbers of variants of a sequence, and then screens these variants to include only those that have a desired property. Directed evolution is most easily applied to the creation of optical reporter genes because the fitness of the mutation is directly measurable via the reporter function.

Using directed evolution to create a light responsive DNA synthesis protein is more difficult. If one had a partially functional form, then the cells could be directed to write out the sequence for an antibiotic resistance gene (e.g. kanR). In this case, only the cells that correctly interpreted the light signals would survive. A simpler variation of this approach could to direct the organism to synthesize a shorter sequence such as a microRNA that would protect the cell.

Mechanistically, the light directed DNA synthesis protein might operate like a cylindrical typewriter shown in Figure 2. In this model, the protein complex would condense around a recognition sequence of a small blank plasmid. Once condensed, the complex would break the plasmid and passively bind dATP, dGTP, dCTP, and dTTP. Upon exposure to a specific wavelength of light, one of the four nucleotide carrying arms would bend to bring either A, T, G, or C into the reaction core. When in the reaction core, the base would be added to one of the cut ends of the plasmid. When the light is turned off, the arm would relax, and in doing so allow it to be recharged with another nucleotide base. At the same time the newly added DNA base would be ratcheted forward and the complementary base added. By repeating this process of light/dark illumination one could then encode any DNA sequence. At the end, a separate light signal could be provided to cause the complex to dissociate and release the plasmid.

The original blank plasmid could then be recovered by a different set of proteins as shown in Figure 3. This recovery set of proteins could bind to different ends of the plasmid core and carry out a crossover reaction to recover the original blank plasmid along with a plasmid containing only the synthetic sequence. This same mechanism could be used to create more blank plasmids by including the two recognition sequences (shown as black and white rectangles) in the synthetic sequence.

Light Directed Remote Control

A second requirement for the SBM is the ability to use light to direct the resident organism to move. Phototaxis is already common among many organisms, and as such could be harnessed.

One way to build such a resident organism would be to use a variant of directed evolution I am calling “The Square Dance of Death” shown in Figure 4. In this approach, some of the organisms are told to become motile and move to a new spot. Those who comply survive, while those that do not comply are killed by ultraviolet light. By repeatedly migrating the organism to different locations, one should select for an increasingly light responsive population.

Resident Organism Selection

For the SBM to function, it needs a base organism. For operational simplicity, this organism must have the following properties:

Motility: Allows data transfer and maintenance operations

Allows data transfer and maintenance operations Asexual and sexual reproduction : Aids in evolution

: Aids in evolution Ability to fix nitrogen : Allows closed cycle life

: Allows closed cycle life Ability to photosynthesize : Allows closed cycle life with only light as an energy source

: Allows closed cycle life with only light as an energy source Rapid growth : Resident organism growth defines the SBM operating speed.

: Resident organism growth defines the SBM operating speed. Sequenced genome: A sequenced genome allows the user to rationally design parts that will correctly interact with the resident organism.

Given these requirements, members of the bacterial phylum cyanobacteria (see Figure 5) are a clear choice. Cyanobacteria or “blue-green algae” are unusual in that they are both photosynthetic and able to fix nitrogen.

Synechocystis sp. PCC 6803 has been fully sequenced and is relatively well studied (1257 publications found on pubmed). Doubling time is on the order of 12 hours, so growth is relatively slow. This species has been shown to migrate toward and away from different light sources 12010493)

Anabaena variabilis has been fully sequenced and is relatively well studied (424 publications found on pubmed) Doubling time is on the order of 14-24 hours, so is relatively slow.

With a SBM like the one describe here, it would provide multiplex automated genome engineering (MAGE) (19633652) capabilities for a wide variety of users. Because it is self-contained, it could be operated with a minimum of laboratory equipment and little to no wet laboratory experience. To many experimentalists the idea of a SBM may sound crazy, but a similar idea sounded crazy to the first computer engineers before the PC.

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