SURF Proposal -2013 Synthetic biological circuit 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 plac/ara-1 [11] which is composed of the activator operator site from the araBAD promoter and a repressor

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.

J Stricker et al. Nature 000, 1-4 (2008) doi:10.1038/nature07389

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.

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, as proposed 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-vivo system, we would shift to the TX-TL invitro 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-Bennett circuit, 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.

Proposed Timeline
(Week 1-2) - Working on the 1st part of the project i.e. plasmid preparation, transformation and 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 cell free expression system. 1st run on the in-vitro platform. (Week 4-7) Test various conditions for expression. Tune parameters such as rate of degradation 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 achieve any 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 system out 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 cell dilution. 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.

References
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