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.
within a new field
biology, which seeks
to apply engineering
techniques to build
to perform functions
in medicine, the
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