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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).