Biology Reference
In-Depth Information
of
100 extra nucleotides surrounding the functional motif resulted in a promoter library
that showed less variation than standard promoters across different contexts.
For instance, promoters from this set, when placed downstream of UP sequences known
to enhance transcriptional activity, were largely insulated from additional stimulation.
Therefore, the combination of these insulated promoters with the more standard
inclusion of transcriptional terminators upstream of promoters to prevent residual
transcription from nearby promoters should allow the part to become more robust to
genetic context.
B
If the mechanism of regulation permits, insulation can be made more definite by the
insertion of ribozyme sequences between regulatory parts.
14
For example, Lucks et al.
wished to create an RNA regulatory cascade where an antisense RNA (A1) would attenuate
the expression of a second antisense RNA (A2) under the control of a
cis
-RNA attenuator
(AT1) that sensed A1.
24
This involved the fusion of AT1 to A2 such that A1 would regulate
the production of A2. However, once generated, A2
s function is significantly diminished
if AT1 is attached. Therefore, a self-cleaving hammerhead ribozyme sequence was inserted
between AT1 and A2 such that once A2 was produced it would be cleaved from AT1.
In short, the part, A2, was made more robust through the insertion of a ribozyme
sequence that physically cleaved A2 from a destructive context.
'
Recently, Qi et al. developed a more general approach to regulatory part cleavage from
physical context. This strategy involves the insertion of CRISPR repeats between parts such
that once transcribed, a coexpressed Csy4 protein cleaves the parts from each other.
13
This
allows the generation of homogenous 5
-UTRs regardless of promoter (which often have
ill-defined transcriptional start sites), and the separation of multigene operons into separate
mRNAs for each gene. It also allows multiple
cis
-regulatory RNA parts, such as riboswitches
that control transcriptional continuation, to be reliably fused in tandem, since CRISPR
cleavage will ensure that each subsequent
cis
-regulatory RNA part is cleaved from the
previous ones before it exerts its regulatory control, mitigating possible interference from
upstream mRNA structure on regulatory function. Using this CRISPR strategy, they
demonstrated dramatic decreases in part variation across a number of operon assembly
studies. In addition, this strategy was essential to the recent construction of multi-input
NOR gates through the fusion of up to four mutually orthogonal RNA transcriptional
attenuators in series, as each attenuator required a free 5
'
71
-terminus for function.
45
'
Finally, if one moves beyond the scale of intracellular circuits to intercellular circuits,
one can rely on the physical separation between cells for insulation of part behavior from
unpredicted compositional effects. For example, recent work by Tamsir et al. shows that
cells containing single NOR logic gates, consisting of inducible promoters, some of which
respond to quorum molecules and output quorum molecules, can be spatially patterned
such that computation progresses from one manually spotted location of cells to the next
(for example, from left to right on solid plates).
15
Since cells sense and output different
quorum molecules, and since quorum molecules become dilute as they diffuse away from
their source, the arrangement of different spots creates different logics that can be integrated
in a final spot which outputs a signal according to desired logic functions. In fact, all
16 possible two-input logic gates were constructed from different arrangements of
NOR gates encapsulated within whole cells.
The key point of insulation is to remove unwanted interactions and uncertainty from the
operation of standard biological parts. Ideally, the parts above would have characterized
function that was largely invariable or at least predictable across different immediate genetic
contexts, host cells, and environments. There remains a fair amount of low hanging fruit in
this area: methods for minimizing unwanted physical interaction among synthetic and host
elements; methods for reducing the energetic load of designs on the host cells; methods that
reduce competition for common components of the circuit (fan-out issues/retroactivity).
