For more information about Synthetic Biology visit the Synthetic
Biology web pages at MIT.
For more information about Biobricks visit the Biobricks
For more information about the repressilator
system see this article.
To improve upon Elowitz's repressilator. We chose to implement
this improvement by decreasing the period using an anti-sense
RNA interference step. The Interfering Repressilator is designed
to exhibit oscillatory behavior in the form of periodic expression
of Yellow Fluorescent Protein (YFP).
Description of System
The success of this system clearly rests on the ability to effectively
and specifically target mRNA transcripts for degradation using anti-sense
RNA. While many papers, articles, and books have been written on the
subject, there are no consensus anti-sense building strategies presented.
We thus chose to implement three different types of antisense inhibition:
KISS, micRNA, and IS10. In the description that follows, the following
nomenclature will be used:
target- the mRNA transcript that we wish to inhibit.
anti-sense- the anti-sense molecule which will bind and
KISS (Keep it SImple, Silly)
The simplest of the three methods, this type relies on a
single-stranded linear 103 bp anti-sense that is specific
to the target of interest. In addition, the first 76 base
pairs of the cI region of BBa_I1010 have been codon-modified
to give a different sequence that codes for the same cI protein
(See BBa_I1030 and I1040).
BBa_I1011 contains the reverse complement of the RBS, start
codon, and 76 bp region for BBa_I1010. Thus, if both BBa_I1010
and BBa_I1011 are transcribed, the transcripts will bind to
each other and BBa_I1010 will not be translated.
Note that BBa_I1010 already contains a regulatory region,
RBS, and coding region (a terminator must be added), while
BBa_I1011 does not - thus, when using this component, the
appropriate regulatory region, RBS, and terminator must be
added to this part.
This anti-sense mechanism relies on two stem loops flanking
an anti-sense sequence that is specific for the target. The
function of the stem loops is to maintain the anti-sense region
in a quasi-linear state. BBa_I1012 is built in this manner,
with a linear region that will bind over the RBS, start codon,
and 76 bp of BBa_I1010.
This method is modeled after the mechanism by which IS10
inhibits production of IS10 transposase. The anti-sense strand
is transcribed from the complementary strand of the target
(see below), resulting in an anti-sense strand that is 115
bp long, of which 35 bp are complementary to the target. In
the absense of a target, these 35 bp form a weak stem loop
with the rest of the anti-sense molecule (see below). The
key element of the system is the loop at the tip of this stem
loop (C-G-G-C-U-U...), which is held in a linear state by
the rest of the loop. Upon exposure to the target, the linear
loop is able to bind to the 5' end of the target (G-C-C-G-T-T...),
and initiate an energetically-favorable zipping/twisting-together
of the target and the 5' end of the stem loop (see below).
In other words, one side of the weakly stable anti-sense stem
loop binds 35 bp of the target, to form a more stable duplex.
I1010 and I1013
Biobricks part BBa_I1013 codes for the exact anti-sense stem
loop used in IS10, with two base changes. The 5'-most residues
from IS10 anti-sense transcript ( U-C), which do not form
part of the stem loop, were changed to G-A. These two bases
are reverse-complementary to the first two base pairs of the
wildtype cI coding region of BBa_I1010, and thus can bind
this region. The rest of the stem loop is wild-type.
The BBa_1010 transcript is targeted by BBa_I1013. The first
35 bases at the 5' end of BBa_I1010 are identical to the first
35 bases at the 5' end of the wild type target, with two differences.
Note that three bases T-G-C (which code for cysteine) have
been inserted at the 5' end of the cI coding region immediately
after the start codon. This allows us to use a wild-type binding
pattern at the base of the stem. Since this cysteine is added
to the N-terminus of cI, it is not expected to alter the repression
ability of cI.
We simulated our model using Matlab. Differential equations were developed to model the change in concentration of 5 key species: cI mRNA, lacI mRNA, anti-sense mRNA (asRNA), cI protein, and lacI protein. The model uses the parameters from Elowitz (2000). The only other parameter that was required was the rate constant for mRNA/asRNA binding. This rate constant was about 1E6 M-1s-1 (Jain, 1997), which translates to 2E4 molecule-1s-1.
It doesn't oscillate. That's it. Unfortunately, it was determined that our design is very sensitive to the rate constant for mRNA/asRNA binding. If this constant is too low (ie below 0.02 molecule-1s-1) then the system fails to oscillate for the values of the other constants as given by Elowitz. With the constant at its expected value (two orders of magnitude below this critical value) the system fails to oscillate for all values of the other constants within several orders of magnitude. It appears that this rate constant is the critical piece in our design, and we were not able to come up with strategies to get around this problem.
Main Program Code
Important Parameters (and Links to Articles from which
As described in the summary section, the critical parameter is the rate constant for mRNA/asRNA binding. It appears to limit the performance of our design, for which no other reasonable range of values of the other constants could compensate.
Challenges and Debug Plan
(Why it may or may not work) BlahBlahBlahBlahBlahBlah
Debug Plan to make it work BlahBlahBlahBlahBlahBlah
Comments for future projects groups
synthetic oscillatory network of transcriptional regulators, Elowitz
M.B. , Leibler S., Nature(403),335-38: 2000
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Coleman, J., et al. Cell (1984) 37, 429-36.
E. coli codon usage table (http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html).
Jain, C. (1995). IS10 Antisense Control in Vivo is Affected by Mutations Throughout
the Region of Complementarity Between the Interacting RNAs. J. Mol. Biol.
Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo.
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Kittle, J.D., Simons, R.W., Lee, J., and Kleckner, N. (1989). Insertion Sequence
IS10 Anti-sense Pairing Initiates by an Interaction Between the 5' End of
the Target RNA and a Loop in the Anti-sense RNA. J. Mol. Biol. 210:561-572.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210
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