Method

For this project, we focused on the use of invertases as our recombinase inputs. By orienting two identical copies of an att site in opposite directions, the corresponding invertase which recognizes these att sites will effectively flip the entire duplex region located between the sites. Using this property, we developed two separate algorithms which place at sites intelligently between and around the genes of interest, such that all of the desired combinations and permutations can be achieved through these enzymatic inversions.

The output of our Pancake and LinkSort translations is a large composite part-containing set of genes with att sites properly inserted such that all permutations and combinations of the genes can be achieved given a particular set of invertase inputs. However, we realize that the user is most likely not interested in the entire combination/permutation output space. Due to this, we propose a top-down design approach to a tailored reconfigurable circuit.

If the user is not interested in the entire permutation/combination space, many of the att sites can be removed from the fully reconfigurable design. Thus, Trumpet follows the following process: First, the user specifies the permutations/combinations of interest. Then, based on which algorithm is chosen (Pancake or LinkSort), a fully reconfigurable circuit is produced with att sites that allow for all permutations and combinations. Lastly, all of the att sites which are unnecessary for achieving the desired permutations and combinations are removed. The output of this is a tailored design with only the att sites necessary for the user’s desired permutations/combinations. We call this entire process the top-down design approach.

We hope that by attempting to solve this problem, we might inspire others out there to design their own att site placing algorithms which are more efficient than ours. Or, perhaps, people interested in this problem can find new and exciting ways to reconfigure circuit plasmids. We believe that if this problem could be solved very efficiently, it might pave the way to more interesting genetic circuits.

Design Example

To the right, we see a simple, yet nontrivial, solution for a two part design which helps illustrate the use of invertases for re configuring a plasmid. This circuit plasmid allows for all permutations/combinations of the two genes using only three sets of invertase sites, illustrated by the sets of red, blue, and orange triangles. By flanking each gene with a its own set of att sites, each gene can be turned "on" and "off" by inputting the corresponding invertase. To change the order of the two genes, the invertase corresponding to the outermost att sites (the red sites) must be inputted into the system. The cartoon to the right displays a sequence of invertase inputs, and the effect each input has on the circuit plasmid. The order of these inputs does not particularly matter in this example; however, in more complicated designs the sequence is very important. Also, it should be noted that only four out of the six possible configurations of this design are illustrated.

Demo and Download

Results

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Future Work

• A surface application for Trumpet is still being developed. On the surface, users would be able to directly manipulate and arrange parts and participate in collaborative design and decision making. Design prototypes include integration with the Clotho database and ability to drag and drop components from a toolbox of parts. The patterns generated by Trumpet could then be translated for use in the lab with Puppetshow and further integration with other applications.
• The two algorithms we developed have different required invertase complexities which scale with the number of genes,n. The "better" of these two algorithms has an O(n²) complexity. We believe that lower complexity algorithms may exist. Future versions of Trumpet will implement more efficient att site placing algorithms, possibly using a bottom-up approach rather than a top-down approach.
• Each of Trumpet's algorithms has an associated key generator. For any permutation, the key generator returns the proper key according to the logic behind the algorithm. While the keys returned are certainly correct, they are not guaranteed to be the shortest possible keys. As the complexity scales with n², we can imagine that some invertase sites which were included in the design for permutation A, for example, could also be used for permutation B. Unfortunately, the key generator has no way of knowing that these sites would also be useful for permutation B. Intelligent algorithms should be developed to find these minimum length keys.
• Future versions of Trumpet should also allow the user to specify the maximum length of the longest key, as this may directly correspond to the number of vectors added to the cell. By specifying this length, future Trumpet could return a set of designs with this maximum key length. As a set, these designs still achieve all desired permutations/combinations. Our current version of Trumpet allows the user to split a design into small sub-designs in an attempt to address this feature.