Applications Link; This link will go to a common page for all
groups describing why we did these projects. (Do not work on this for each
group, we'll take care of it.
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 build an oscillator out of genetic components that exhibits better
autocorrelation than the repressilator.
Description of SystemWe
designed 3 new repressor-promoter pairs (all from the lamboid cI family
- 434 cI, P22 c2, and HK022) in order to make a double oscillator - two
linked repressilators, each controlling its own reporter (HK022 was chosen
to be our 'spare part'). Theoretically, the two rings will synchronize
each other and stabilize the oscillations of the reporters over time.
Theorized Biological Mechanism
- Base Plasmid for Structured Assembly with link to Actual Biobricks
- Transcriptional Terminator 2
- TE Transcriptional Terminator from Bacteriophage T7
- RBS-5 Elowitz RBS
- LacI Protein LVA w/o RBS
- TetR coding region LVA
- cI coding region from HK022cI- LVA
- cI coding region from Lambda - LVA
- cI coding region from 434
- c2 coding region from p22 -LVA
- CFP w/o RBS w/ LVA
- YFP w/o RBS w/LVA
- IAP Inverting regulator driven by LacI (BBa_C0010, 11)
- Inverting regulator driven by C0040 (TetR)
- cI Regulatory Region
- cI regulator from Lambda
- Regulator region driven by C0052 (cI)
- Regulator region driven by C0053 (c2)
We performed several simulations using Matlab (Continuous) and
Stochastirator (Discreet) to test the expected performance of our
system. The results and setup is discussed below.
As discussed above, the system is setup as a a double oscillator,
with each system sharing a common protein. Since there were six
proteins to choose from, and five positions in which they could be
located, more than 7000 permutations are possible. To choose the best
performing systems it was necessary to simulate each permutation in a
systematic manner and order each system according to some ranking
values, such as stability of phase, frequency, and amplitude.
Link to Graphs here.
The Source Code of the Scripts is above.
Important Parameters (and Links to Articles from which
Initially, the Elowitz RBS (BBa_B0034)
will be used with all coding sequence components. This RBS can
be adjusted if necessary based on results from debugging experiments
- RBS-1 Strong, BBa_B0031
- RBS-2 Medium,
BBa_B0032 - RBS-3 Weak, BBa_B0033
To ensure termination of transcription, a double terminator will be
used after every coding sequence (BBa_B0012
- TE Transcriptional Terminator from Bacteriophage T7
followed by BBa_B0011
- Transcriptional Terminator 2).
Since the phage repressor regulatory regions include partial promoter
sequences driving transcription on the reverse strand, a bidirectional
terminator will be placed upstream to prevent undesirable transcription
HK022 was chosen as the spare part for two reasons: one, the least
is currently known about its possible interactions with other phage
repressor/promoters, and two, it has a significantly higher degree
of cooperativity than the other lamboid repressors, so it is most
likely to unbalance the rings.
Another design issue is plasmid copy number: we are placing our system
on low-copy plasmids, but the reporters on high-copy in order to create
stronger YFP and CFP signals. This design may result in over-dilution
of repressor proteins (tetR and LacI, specifically), however.
Challenges and Debug Plan
1. Cross-talk between different repressor-promoter pairs.
This may lead to either stable or chaotic behavior (i.e. not oscillatory).
2. The different repressor-promoter pairs are imbalanced with
respect to either repressor or promoter strength. According
to simulations, this may lead the two oscillators to have sufficiently
different periods such that synchronization is impossible.
1. Cross-talk experiments
Place each repressor under the control of an externally-controllable
promoter (such as LacI or tetR) and each regulatory region in control
of YFP. Each repressor and regulatory region can be tested in
a combinatorial pair-wise fashion to assess both cross-talk and the
tightness of repression (if the repressor is used in conjunction with
its corresponding regulatory region). These experiments have
the additional benefit of establishing transfer curves for each repressor-promoter
pair. This leads to a total of 36 parallel experiments.
2. Balance experiments
Much of the information concerning the relative strengths of the
promoter-repressor pairs can be established in the experiment detailed
above. However, an additional debugging strategy may be to take
the repressilator and swap each of the three new phage repressor-promoter
pairs for the lambda cI repressor-promoter pair to establish whether
there is oscillation and if so, how its frequency compares to the
repressilator. The same experiment can be repeated in a phage-derived
repressilator to compare lacI and tetR.
3. Real-Time Debug
Since the double oscillator is set up so that tetR and LacI are in
separate rings, each ring can be separately disabled at any time during
the experiments with either IPTG or ATC, if necessary.
An interesting idea to pursue is to make the repressilator with a
protease inserted at one step in addition to the repressor.
The corresponding component in the repressilator could be degraded
specifically by this protease in order to achieve a smaller period.
The TEV protease is an ideal candidate for such a project.
Usual reference format. If possible include description of each
article and link to PDF file in your group folder.
Group Members (Include Picture of Group)
Jose J Pacheco