Biology Reference
In-Depth Information
An experimental implementation of a two invertase, Full
Rank System: Basic Design and Construction of the Device
To begin to understand the physical constraints on the building of invertases-
based memory devices and switching elements, we chose to construct a device
like that shown in Figure 1C. While we could have designed for states 1-4 of our
device each to have a separate active output, to simplify our design and to make
the measurements realizable we settled on a design with fluorescent proteins to
the left and right of the device such that only states 3 and 4 would show output.
However, as will be discussed below, the invertases we chose show reversible flip-
ping and so the fifth state mentioned above also becomes accessible. If seen, this
state would output both the left and right fluorescent proteins.
The hin and fim systems were chosen for constructing this new genetic switch
for the following reasons, in addition to the ones that are obtained for any recom-
bination circuit outlined above. 1) They have very specific recognition sites. These
sites are well known and their DNA-protein interactions have been described. 2)
They are independent of each other. Unlike inversion recombination systems that
are from the same family, the hin and the fim systems are completely orthogonal
with different mechanisms. 3) They are inducible. Only in the presence of the
Hin and FimB proteins can the system invert. 4) Their mechanisms have been
well studied. There is some twenty years of literature exploring the recombination
mechanisms. 5) They are known to be flexible. The distance between the recom-
bination sites can be varied greatly, from a few hundred base pairs to several kilo-
bases. This allows the possibility of adding additional promoters or genes in be-
tween the recombination sites. 6) They have very low rates of excision, even when
the inversion sites are arranged in direct repeats. Other inversion recombination
systems (for example Cre/Lox) will excise the region between direct repeats.
Each inversion reaction was initiated by the expression of one of the recombi-
nases, FimB or Hin. The gene encoding FimB was expressed from the arabinose-
inducible araBAD promoter (PBAD) [26], and the gene encoding Hin was ex-
pressed from the aTc-inducible tet promoter (PTet). Fim and Hin are expressed
from a plasmid (pZB) containing the two promoters [27]. The recombinase genes
were harbored on a separate plasmid from the switch to facilitate testing. Thus,
the system has two inputs and two outputs. Although the two recombinases were
expressed from inducible promoters, it would be possible to express them from a
different input, for example environmental or metabolic sensors.
The target DNA regions were harbored on a separate plasmid. The switch
region was synthesized de-novo and cloned into pPROBE-gfp. The red fluores-
cence gene was cloned in last. The fluorescent proteins GFP and RFP are placed
to the left and right of the device respectively. The resulting plasmid is multicopy,
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