Figure 4: One of several of the most efficient ways to convert BAC construct into ABC construct is to use successive flips of hix-flanked segments of the DNA.

“Undergraduate
students worked
within a new field
called synthetic
biology, which seeks
to apply engineering
principles,
mathematical
modeling, and
molecular biology
techniques to build
biological machines
to perform functions
with applications
in medicine, the
environment, energy,
and technology.”

tion would be manipulated within the bacterial cells. In nature, Salmonella typhimurium has the ability to invert a segment of its DNA to switch from one gene to another so its extracellular surface is covered by a different protein. The cell’s mechanism, called the Hin-hix inverter, was reconstituted by a previous iGEM team from Missouri Western State University and Davidson College for use in E. coli to solve the Burnt Pancake Problem [ 2]. Each segment that we want to flip is flanked by a 26-basepair hix site on each side. In the presence of Hin proteins, the DNA is temporarily cut at a pair of hix sites, inverted, and reattached. To simplify our notation we used a prime (’) to indicate that a segment of DNA was flipped from its original 5’ to 3’ orientation. In Figure 3, the yellow triangles represent the hix sites. Figure 4 shows one series of successive inversions that converts the test construct in Figure 3b to the solution construct shown in Figure 3a.

Once the problem was encoded in
DNA we benefited from the greatest
advantage of bacterial computing. A
single E. coli bacterium will replicate
itself approximately every 30 minutes.
Growing a bacterium overnight results
in 2^ 30 (approximately a billion), identi-
cal, independent biological-processors.
When exposed to the Hin protein each
E. coli cell flips some segment of its DNA
flanked by the hix sites. Even if this pro-
cess is random (which is still an open
question) the probability is essentially
1 that at least one in a billion biological
processors will find the solution.

HOW THE BACTERIA RESPONDS

The final design challenge was how to know which cells have solved the HPP.

A bacterium with Edge A in the first position in 5’ to 3’ orientation will express red fluorescent protein because the two halves of the RFP gene have been successfully united and thus appear red under ultraviolet light. If in addition Edge B occurs in the second position, the two pieces of the GFP will be reunited and the bacterium will additionally glow green. The presence of both red and green fluorescent protein indicates a solution to our HPP, a combination that appears yellow under ultraviolet light.

We created several beginning constructs for our HPP problem. One of our constructs, corresponding to the schematic in Figure 3b, began with no fluorescence. But individual clones gained fluorescence after Hin-mediat-ed flipping of the DNA.

Figure 5 shows photographs of plates from the lab. Figure 5a shows uncolored bacteria with no Hin introduced, where as Figure 5b shows a similar plate that began with all bacteria uncolored, but changed to colored after the introduction of Hin. The bacteria that glowed red are those that managed to flip their DNA at the hix sites