The Bacterial Full Adder

Abstract The Full Adder project seeks to create bacteria that can add two one-bit numbers. Such an organism would be the biological equivalent of a digital full adder, which is a fundamental building block of complex electronic circuits. Since many teams have difficulty creating even simple circuits like XOR gates, this project would seem nearly impossible. However, we have found that the problem can be greatly simplified if we use a simple “encoding” on the outputs of the full adder. Subsequently designing a second circuit to “decode” the output is theoretically significantly easier than creating a complete full adder outright. Our insights may prove useful to any genetic engineer or synthetic biologist working on highly complex systems.

General Overview
Engineering Problems Construction of a bacterial full adder faces two immediate obstacles. First, an electrical full adder requires at least 5 logic gates. However, a single AND gate in bacteria may require as many as 11 BioBricks (as shown by the Peking team in 2009). Chaining together tens of BioBricks would be very difficult and may result in plasmids that are difficult to transform. Second, many of the current “logic gate” circuits are not easily connected to each other. For example, the ETH 2006 XOR gate can not be connected to the Davidson-Missouri Western (DMW) 2008 XOR gate since the inputs and outputs are not the same; ETH has PoPS (Polymerase Per Second) inputs and output, but DMW uses various chemicals. Simply using PoPS-only parts may be an attractive solution, but that may exacerbate the first problem as parts with PoPS inputs and outputs tend to be more complex. Furthermore, there is a significant possibility of some PoPS parts interfering with the operation of other PoPS parts as these are huge, complex circuits, many of which share some parts. Insights We had two major insights into the obstacles to create the Full Adder. First, we developed a method to create biological AND gates. Second, we found that, if we change the output of the full adder from two outputs to three outputs (our so-called encoding), we can create the circuit with four AND gates. Of course, this is no longer a faithful rendition of a full adder. However, the methods we used to create the AND gates should be helpful in the creation of a whole new generation of fusion proteins, and it should be easier to create another circuit that converts our three output adder to a standard two output adder than creating a single monolithic full adder circuit.

Peking 2009 AND Gate
Light Response One AND gate that we decided to use was created previously by the Peking 2009 iGEM team. It relies on T7ptag, T7 polymerase with two early amber mutations (stop codons) such that normal translation will result in a truncated non-functional protein, and SupD, a tRNA which allows for translation through amber stop codons (an amber suppressor). If just T7ptag or just SupD is expressed, there will not be any functional T7 polymerase, but if both T7ptag and SupD are expressed, then functional T7 polymerase will be created. To complete the gate, we place T7ptag on one inducible promoter (our first input), SupD on a different inducible promoter (our second input), and our output on a T7 promoter. Note that this system relies on the fact that basal transcription from the T7 promoter is extremely low, but once functional T7 polymerase is present, transcription is very high. This new bond distorts the conformation of the protein, causing the detachment and unfolding of the Ja-helix (see figure 1). In natural AsLOV2, the unfolding of the Ja-helix results in further downstream signalling. However, we will be most interested in the fact that the Ja-helix detaches when LOV2 is hit by blue light.

Split Fluorescent Proteins

Overview In our search for AND gates, we sought AND gates that functioned similarly to Peking’s AND gate (and for that matter, most other AND gates). These often consist of two components that are individually non-functional, but are functional when both are expressed together. Ghosh et al. created exactly that with their split GFP. They cut GFP, which usually displays green fluorescence, into two parts, each of which did not fluoresce. Then they fused leucine zipper domains to each part, which allowed them to reconstitute functional GFP when both were expressed together. They dubbed the two parts nzGFP and czGFP. Split fluorescent proteins are particularly attractive in C. elegans as they allow for combinatorial marking of cells. As C. elegans are transparent, a common method to study particular cells is to put fluorescent proteins under the control of promoters specific to those cells. Thus, only the interesting cells light up under a fluorescent microscope. However, some groups of cells are difficult to target with just a single promoter. With split fluorescent proteins, a scientist can light up cells which have high transcription at two different promoters, allowing for much a finer selection of cells to study. Thus, further research on split fluorescent proteins was done in C. elegans (Ghosh et al.’s study was in E. coli). Zhang et al. created similar split CFP and YFP (cyan and yellow fluorescent proteins). All of these split fluorescent proteins function essentially as AND gates. The inputs are activators of our choice of promoters we place the split fluorescent proteins under and the output is fluorescence. The primary advantage of this system is its simplicity. It just requires two proteins and two coding sequences. Furthermore, the coding sequences do not code for enzymes which can have all sorts of messy interactions with other parts. The primary disadvantage of this system is that the output is limited to fluorescence.   Construction As mentioned previously, a significant amount of the research on these split fluorescent proteins has been done in C. elegans. Unfortunately, C. elegans requires introns for the expression of certain genes, and so much of the easily accessible DNA with split fluorescent proteins contains artificial introns, which would mess up expression in E. coli. We therefore decided to recreate split GFP, CFP, and YFP using fresh GFP, CFP, and YFP from the parts registry. However, we aimed higher still: by cloning the zipper portions with the easily fusable RFC25 prefix and suffix to create new BioBricks, we paved the way for future split protein construction. As demonstrated by our circuit, split proteins have the potential to greatly simplify genetic circuits. First, we cloned the zipper domains from nzGFP and czGFP with the RFC25 prefix and suffix, which allows for easy in-frame fusion of coding sequences. Then we mutagenized GFP (BBa_E0040) so that it contained the necessary restriction sites in the correct positions in order to create nzGFP and czGFP. Finally, we cut and ligated the parts. This method is very general and should be easy to apply in searches for other split proteins.

Bacterial Full Adder Circuit
Logic Circuit Our circuit fulfills full adder functions by taking a biological input representative of a binary input and giving a biological output that is representative of a binary output.

Results
Overview Unfortunately, we were unable to finish constructing our circuit in time for the wiki freeze. The following parts have been submitted to the Registry of Standard Biological Parts.