Figure 2: The three-node directed graph on which proof-of-concept bacterial computer experiments were conducted is shown. The unique Hamiltonian path in this graph is AB.

 

Our first design consideration was to determine how the problem could be encoded into DNA. DNA is endowed with a natural directionality, known as 5’ to 3’, which we exploited to represent the directed edges of our graph. DNA components must be present in the correct 5’ to 3’ orientation and spatial order to make a functional gene capable of producing a protein. A gene functions when there is a promoter, a ribosome binding site (RBS), and a coding sequence for a particular gene. A transcriptional terminator may be included at the 3’ end to prevent the gene expression machinery from continuing farther down the DNA.

The critical step for coding an HPP was splitting the genes that produce red fluorescent protein (RFP) and green fluorescent protein (GFP) into t wo pieces, and inserting a short segment of DNA between the two pieces. Fluorescent protein genes are standard reporters in the synthetic biology community because they cause cells to glow, allowing researchers to monitor the results of an experiment in real time, without isolating the DNA. These reporters and others are made available to the iGEM community through the Registry of Standard Biological Parts [ 3].

Finding appropriate locations to split these genes was a significant bioinformatics challenge. The insertion location had to be chosen so that the split gene was still functional, but a portion of RFP combined with a portion of GFP would result in a non-func-tional protein. To encode the HPP in a bacterial cell, we split the RFP into two pieces, RFP1 and RFP2, and the GFP into two pieces, GFP1 and GFP2. The three edges of our graph were built by taking the second portion (the 3’ piece) of the source node followed by the first half of the destination node. More specifically, edge A consisted of RFP2 and GFP1, edge B consisted of GFP2 and the transcriptional terminator, and edge C consisted of RFP2 and the transcriptional terminator. Our control construct ABC and one of our test constructs, BAC, are shown in Figure 3.

Our second design challenge was to determine how the DNA informa-

“Many other
iGEM projects
have components
of computation,
simulation, and
mathematical
modeling. The
results have
application in
medicine, the
environment, and
biofuels, as well as
other benefits from
data analysis and
model building,
to the testing of
experimental
designs.”

Figure 3: (a) A blueprint for the control construct ABC is contrasted with (b) the blueprint for the experimental construct BAC. a

b