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 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 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 eight possible configurations of this design are displayed in the cartoon.

Tutorial and Download

Tabletop Implementation

After seeing the success of the desktop implementation of Trumpet, we realized it could be useful to have a tabletop version of Trumpet to allow for collaboration between users. The process of choosing specific combinations of genes, promoters, ribosome binding sites, and other genomic sequences inherently relies on collaboration, since the outcome of such a process will influence the work that each member of the lab needs to complete. Different members of the lab will have different research goals for their work, and the best way to figure out how each person will accomplish their goals in the context of the overall research question requires input from the entire team. A tabletop Trumpet application will allow the entire lab to get involved in determining research goals.

To this end, we created and iterated upon a design for a tabletop version of Trumpet. After some initial discussions in the computational team, we showed our goals to the wetlab researchers and got their feedback on which features they would find useful in a tabletop version. While we were not able to create the tabletop version of Trumpet this year due to time constraints, we anticipate adding it to the suite of Surface tools in the future.

Results

After designing the primers for the TB transcription factors and sequencing these genes, we can build our constructs with the genes of interest and invertase sites using Trumpet's invertase tree. The tree helps us visualize when we need to apply each invertase to the genetic device. We will be using PCR-based methodologies to add the invertase sites and the transcription factors to a biobrick backbone. This way we can restriction digest these parts and ligate them to the basic devices we constructed.

In order for the correct permutation to take place, we need to treat the complete construct with invertases. The invertase tree will be useful in telling us the order in which we need to treat the device with the appropriate invertases. The first invertase will be on a seperate plasmid then during transformation of the larger construct we will heat shock the construct to prompt the invertase's actions on the construct. This step is will activate the inversion and flipping action of the desired permutation. With these steps, we will be able to manipulate the actions of the promoter on a gene or even investigate the relationships between a variety of TB transcription factors in a network.