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SURF Proposal -2013
Synthetic biologicalcircuit design implementing protein degradation in-vitro
By: Rohit Sharma Mentor: Prof. Richard Murray Co- mentor: Zachary Sun
Introduction
Synthetic biology, supported by advances in genetic engineering, is progressively being used as a
tool to understand and manifest logical forms of cellular control [1]. The successful design and
construction of landmark synthetic gene networks the toggle switch [2] and the repressilator [3]
has enabled modules of increasing complexity. A major bottleneck in implementation, however, has
been the time required to engineer circuits, in-vivo. In-vitro systems [4] shorten engineering time by
eliminating the need to work with intact viable cells. In such systems, cell extract of the host
(typically E.coli) is sufficient enough to obtain genetic expression with high expression efficiency.
Examples of circuits designed in cell free expression systems include bistable switches [5], oscillators
[6] and logic gates [7].
Many circuits, however, do not function like their in-vivo counterparts, in-vitro. The original toggle
switch requires around 3-4 hours to begin switching and over 5 hours to completely change states
[2]. These synthetic modules are regulated by instantaneous concentration of the proteins that act
as circuit activators and repressors. Although the switches may change input states, it takes a lot of
time for the already formed activator/repressor and for the reporters to degrade via dilution due
to cell division, and hence the circuits to actually switch their state. In cell free expression systems,
where intact cells are absent, such dilutions can be mimicked by degrading the protein using
proteases [8]. Under the supervision of Prof. Richard Murray, substantial efforts have been made to
upregulate the expression of AAA ATPases ClpX and ClpP which selectively target proteins with ssrA
degradation tags. Such tag mediated degradation helps in selectively reducing the half life of the
protein of interest.
We propose the in-vitro implementation of a classical Stricker-Cookson-Bennett oscillator [9] in
which activators, repressors and reporters has an ssrA degradation tag. An additional vector, which
gives the constitutive expression of the ClpX and ClpP AAA protease, will enable rapid degradation
emulating cell dilution. We hope to demonstrate a rapid switching in genetic circuits. Similar hybrid
transcriptional/enzymatic circuits allow for state switching over a wider parameter range than
transcription-only circuits [10]. This implies that hybrid systems would likely be easier to tune and be
more robust than systems built only from existing transcriptional components. Demonstrating
protein degradation with the Stricker-Cookson-Bennett oscillator justifies its applications to a variety
of other circuits requiring degradation for dynamics.
Project Description and Approach
We wish to implement Stricker-Cookson-Bennett oscillator on novel cell free platform developed
specifically for synthetic biology experiments. This oscillator typically consists of two loops: a positive
feedback loop and a negative feedback loop. All the circuits have the same hybrid promoter p lac/ara-1
[11] which is composed of the activator operator site from the araBAD promoter and a repressor
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binding site from the lacZYA promoter. Hence the promoter under discussion has a two level control;
it can be activated in the presence of arabinose by an AraC protein and it can be repressed in the
absence of IPTG by a LacI protein. The genetic circuit diagram is given below in Fig. 1. All the
elements namely araC, lacI and deGFP are placed downstream of identical copies of plac/ara-1
promoter. Addition of arabinose and IPTG leads to activation of the promoter leading to
transcription of each of the circuit components. The increased production of AraC in the presence of
arabinose sets up a positive feedback loop. However the simultaneous increase of LacI results in a
negative feedback. The difference between the instantaneous activities of these two proteins gives
rise to an oscillatory behavior.
Figure 1.(a) Placement of various genes in a two loop Stricker-Cookson-Bennett oscillator. (b)
Oscillation obtained in the fluorescence of the reporter GFP in the Hast oscillator
In our setup, we wish to add another vector which contains ClpX protease downstream of a P70
promoter (shown in Fig. 2 (a)). To enable protease specific cleavage, the proteins AraC and LacI will
contain an ssrA tag. GFP does not require any modification because it generally contains such a tag
to decrease its half life. The expression platform which we plan on using is the cell free TX -TL cell
system [12] which is an open source platform with high protein expression not based on T7
reporters. Its functioning requires only the housekeeping machinery to be present in the E. coli
crude extract such as sigma70 and RNA polymerase. These sigma factors are transcription factors
whose presence is necessary for the RNA polymerase to bind to their cognate promoter. Since the
crude extract will contain sigma70, the RNA polymerase can bind to the P70 promoter and a
continuous expression of ClpX can be expected.
J Stricker et al. Nature 000, 1-4 2008
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Figure 2.(a) The ClpX and ClpP protease downstream of P70 promoter. (b) Degradation of
tagged peptides with proteases.
The project will be executed in 3 steps:
In the first part, the in-vivo expression of the Stricker-Cookson-Bennett oscillator, asproposed in literature, will be replicated. We hope to verify the original paper finding as well
as obtain experience in building and testing oscillatory circuits. This work will require
assembly of promoters, genes, and tags and includes plasmid preparation by Gibson
Assembly and cell transformation. We will isolate single cells in microfluidic plates and track
the oscillation of the reporter GFP fluorescence. CellASIC plates keep oscillatory cells in a
single focal plane allowing us to perform high magnification imaging for long durations.
After oscillations have been observed in the in-vivosystem, we would shift to the TX-TL in-vitro platform and include protein degradation. Here the vectors constructed in the first
part need to be modified by inserting the ssrA tags. The protocols for this platform are well
established however we may need do some adjustments depending upon the templates
under study. We will use mathematical models to guide our design and tune parameters to
try to observe oscillations.
Having observed and established protein degradation in the Stricker-Cookson-Bennettcircuit, finally we can then move on to designing and constructing a unique circuit that
implements protein degradation. The exact design of the circuit can be designed during the
course of the project depending upon the experience obtained from handling the oscillator.
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Proposed Timeline
(Week 1-2) - Working on the 1st part of the project i.e. plasmid preparation, transformationand subsequent selection of cells. Isolation to be carried out using CellASIC followed by
microscopic imaging. Establishing the system in vivo.
(Week 3) - Work starts on the 2nd part of the project. Plasmid construction with ssrA tags.Establishing and adjusting the protocol of cellfree expression system. 1
strun on the in-vitro
platform.
(Week 4-7) Test various conditions for expression. Tune parameters such as rate ofdegradation of protein, rate of synthesis of AraC/LacI proteins and test them under different
conditions such as varying arabinose/ IPTG levels or varied temperature etc. This would
require a series of runs and reruns on the TX-TL systems. It is expected that by the end of
June or beginning of July, oscillations can be established in the cell free environment. During
this period, some time will also be devoted to understanding and developing a model that
would help us better regulate the system (Week 7-10) If we are successful in the previous part, the last phase of the project will be
dedicated to developing unique circuit and testing the established in-vitro technique along
with the degradation tags and using the mathematical modeling for predictive purposes
during the course of experimentation.
Possible Roadblocks
Since we are dealing with a complex circuit, it might be possible that we may not achieveany oscillatory behavior on the in-vitro platform. This could happen because parameters
such as protein degradation, rate of synthesis of activator/repressor can push the systemout of the domain where it shows oscillations. This can be taken care of by tuning the
parameters and trying different combinations of regulation. Despite that, if the circuit is not
manifesting oscillations, then we will switch to a simple circuit implementing protein
degradation, such as a switch or oscillator.
We may not be able to implement enough protein degradation to accurately emulate celldilution. In order to realistically implement the oscillator, protein degradation needs to be
increased to a level almost 10-fold above wild type in the in-vitro platform. If we cannot
achieve such an increase in degradation, dynamics in oscillation may be impossible to
achieve.
There may not be enough energy resources to implement an oscillator in the in-vitro system.Even if protein degradation emulates the in-vivo environment, the in-vitro system contains a
limited amount of amino acids, ATP, and nucleotides to sustain function. Because protein
degradation is an energy-intensive process, there may not be enough reserve to run an
oscillator.
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References
1. Khalil, A.S., Collins, J.J. Synthetic biology: applications come of age. Nat. Rev. Genet.11, 367379 (2010).
2. Gardner, T. S., Cantor, C. R. & Collins, J. J. Construction of a genetic toggle switchin Escherichia coli. Nature403, 339342 (2000).
3. Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptionalregulators. Nature 403, 335338 (2000).
4. Buchner, E.Alkoholische gahrung ohne hefezellen. Ber. Chem. Ges. 30, 117124 (1897).5. Kim, J., et. al. Construction of an in-vitro bistable circuit from synthetic transcriptional
switches. Mol. Syst. Biol. 2 (2006).
6. Soloveichik, D., et al. DNA as a universal substrate for chemical kinetics. Proc. Natl. Acad. Sci.USA 107, 53935398 (2010).
7. Takinoue, M., et al. Experiments and simulation models of a basic computation element ofan autonomous molecular computing system. Phys. Rev. E Stat. Nonlinear Soft MatterPhys.78, 041921 (2008).
8. Huang, D., Holtz, J. W., Maharbiz, M., M. A genetic bistable switch utilizing nonlinear proteindegradation.J. Bio. Eng.6, 9 (2012).
9. Stricker, J., Cookson, S., Bennett, MR., Mather, W.H., Tsimring, L.S., Hasty, J. A fast, robust,and tunable synthetic gene oscillator. Nature.456, 516-519 (2008).
10.Shah, N.A., Sarkar, C.A.Robust Network Topologies for Generating Switch-Like CellularResponses. PLoS. Comput. Biol.7, e1002085 (2011).
11.Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichiacoli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res.25,
12031210 (1997).
12.Shin, J., Noireaux, V. An E. coli cell-free expression toolbox: application to synthetic genecircuits and artificial cells.ACS Synth. Biol. 1, 29-41 (2012).
13.Sauer, R., T. et. al. Sculpting the Proteome with AAA+ Proteases and Disassembly Machines.Cell 119, Issue 1 9 18 (2004).