Difference between revisions of "Team:William and Mary/Part Collection"

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Part Collection

The Circuit Control Toolbox

The Circuit Control Toolbox consists of a diverse collection of biological parts that provide precise control over the behavior of any arbitrary genetic circuit. It encompasses a series of components able to achieve a range of modifications; additionally, we provide tools that may be used to measure and characterize the parts within the toolbox itself. The Toolbox contains a total of 118 unique Biobrick parts, many of which have been sequence-confirmed and characterized on multiple BioBrick backbones.

Every part submitted in our Toolbox is flanked by standard Unique Nucleotide Sequence (UNS) segments. These segments, generated by Pam Silver and colleagues at Harvard University, were designed to facilitate insulated assembly of complex and repeat-heavy components without biological interference. They consist of randomized sequences of nucleotides which have been optimized for low hairpin and heterodimer formation frequency; they were further checked to ensure minimal overlap with promoter-like sequences, start codons, and the E. coli genome sequence. By flanking part inserts with distinct UNS sequences, one can achieve secure and efficient synthesis of multiple-gene networks [1].

We chose to standardize each part in our Toolbox with an upstream UNS2 and downstream UNS3 sequence. These sequences were chosen based on their compatibility with standard BioBrick enzymes, as well as the BsmBI enzyme used for Iterative Capped Assembly; UNS1 was thus eliminated from consideration, as it contained the recognition site for BsmBI. By surrounding each part by these two UNS sequences, we were able to make all of our parts far more compatible with Gibson Assembly than the standard Biobrick Prefix/Suffix regions. UNS-guided Gibson Assembly [link to Protocol: UNS-guided Gibson Assembly], using UNS regions directly interior to the Biobrick Prefix and Suffix, provides the advantages of significantly decreased homodimer affinity compared to Gibson Assembly on the Prefix/Suffix regions, while maintaining the 3A assembly capability of the Biobrick standard.

Many of the parts in the Circuit Control Toolbox can be categorized into one of five subsections based on their function. These include the following:

RBS Characterization Constructs

These parts are IPTG-inducible constructs which contain self-cleaving ribozyme RiboJ immediately upstream of the RBS sequence, which insulates the contribution of the RBS to gene expression from contributions from the 5’ UTR region. The inclusion of this element provides an unprecedented avenue for standardizing RBS measurements regardless of associated coding sequences.

Part	Description
K2066034	Promoter and RBS characterization construct – mCherry + Spinach
K2066035	plLac0-1 RBS Characterization Part – B0034
K2066036	plLac0-1 RBS Characterization Part – B0031
K2066044	plLac0-1 RBS Characterization Part – B0029
K2066045	plLac0-1 RBS Characterization Part – B0064
K2066046	plLac0-1 RBS Characterization Part – B0030
K2066047	plLac0-1 RBS Characterization Part – B0035
K2066048	plLac0-1 RBS Characterization Part – B0032
K2066049	plLac0-1 RBS Characterization Part – B0033

2. The inefficiency of the “design-build-test” cycle which is relied upon for the construction of effective circuit models.

The other foundational limitation of genetic circuit construction addresses the inefficiency and unpredictability of the design and construction process itself. The progression from synthesizing parts into a circuit on a plasmid, to transformation and testing in vivo, is a lengthy and expensive process which furthermore is largely variable in terms of actual functionality of the final product [2]. This often leads to a series of trial-and-error testing cycles whose products maintain a persistent level of uncertainty with regard to precise, predictable behavior. Although it is possible to achieve functional genetic circuits in this capacity, greater problems arise regarding the tunability of the product.

The success of any genetic circuit relies on the ability to precisely tune a response to a range of input concentrations; it would therefore be desirable to obtain a reliable method for tuning circuit response, ideally without the need to redesign the core of the circuit. Such a method would allow control over output expression to be implemented in a more rapid and predictable manner [3].

The Circuit Control Toolbox

Our project aims to provide a modular collection of genetic parts which can specifically and predictably tune the behavior of an arbitrary genetic circuit. This collection, which we have dubbed the “Circuit Control Toolbox,” consists of a suite of parts which can be added to the end of a given genetic circuit; each part provides a specific and independently tunable response which allows direct control over the ultimate output behavior of the circuit.

The overall input/output behavior of any genetic circuit can be represented by a graph known as a transfer function, which relates concentration of input molecule to output protein expression. Likewise, any modifications to the circuit affecting input/output behavior can be visualized by a transformation of the transfer function representing the circuit. The Circuit Control Toolbox consists of three distinct tools which prompt unique behavioral changes to the circuit’s output relative to its input, and therefore generate different transformations of the circuit’s original transfer function.

The RBS library provides a collection of ribosome binding sites of varying strength; replacing the RBS within a circuit alters the translational efficiency of the output. This tool effectively allows for scaled changes in the magnitude of a circuit’s output response, thus adjusting the amplitude of the transfer function.

The Decoy Binding Array tool implements molecular titration to tune the circuit’s sensitivity to input concentrations. This modification is accompanied by a shift in the threshold of the circuit’s transfer function.

The Synthetic Enhancer Suite exploits a synthetically modified enhancer/promoter system engineered to allow genetic circuits to generate multi-state responses. In other words, circuits are prompted to produce distinct levels of output based on the concentration of input molecule.This creates a staircase-like curve in the transfer function for the circuit.

Each of these tools functions orthogonally to the activity of the other tools; furthermore, each tool is independently tunable to a specific degree. By implementing and adjusting multiple tools to the desired degree, a diverse range of circuit output behaviors can be achieved, generating a plethora of unique transfer function responses.

Using the Toolbox

The implementation of this Toolbox relies on its generalizability and consistency over any arbitrary genetic circuit. A circuit’s relative output behavior may be influenced by the coding sequence for the output which it controls. In order to ensure that behavior remains consistent across any range of coding sequences, we offer an additional Ribozyme Insulator tool. This ribozyme part, known as RiboJ, insulates a circuit’s promoter activity from the genetic context of the coding sequence, allowing for consistency in the levels of relative expression across multiple coding sequence insertions. The addition of RiboJ as an insulator justifies the application of Toolbox components to the end of any genetic circuit, irrespective of its choice of final output protein [4].

Our Circuit Control Toolbox can easily be implemented in any project concerned with the behavior of genetic circuitry by working through the following sequence of events:

1. Visualize the original behavior of the circuit in question by constructing a characteristic transfer function.

2. Determine the appropriate Toolbox parts-to-use using our mathematical model. This model has been parameterized such that the model parameters correspond to actual physical variables (e.g. number of tetO arrays, plasmid backbone).

3. Swap out the final protein coding sequence in the original circuit with a Ribo-J insulated repressor sequence that is compatible with the Toolbox.

4. Apply the appropriate Toolbox parts to the end of your circuit, and express your original final output protein at the end of the series of Toolbox components.

In this manner, future iGEM teams and synthetic biologists will be able to easily obtain higher levels of precision and control over the behavior of their genetic networks.

References

1. Nielsen, A. A., Segall-Shapiro, T. H., & Voigt, C. A. (2013). Advances in genetic circuit design: Novel biochemistries, deep part mining, and precision gene expression. Current Opinion in Chemical Biology, 17(6), 878-892. doi:http://dx.doi.org/10.1016/j.cbpa.2013.10.003

2. Sun, Z. Z., Yeung, E., Hayes, C. A., Noireaux, V., & Murray, R. M.Linear DNA for rapid prototyping of synthetic biological circuits in an escherichia coli based TX-TL cell-free system. ACS Synthetic Biology, (6), 387. doi:10.1021/sb400131a

3. Lucks, J. B., Qi, L., Whitaker, W. R., & Arkin, A. P. (2008). Toward scalable parts families for predictable design of biological circuits. Current Opinion in Microbiology, 11(6), 567-573. doi:http://dx.doi.org/10.1016/j.mib.2008.10.002

4. Lou, C., Stanton, B., Chen, Y., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, (30), 1137-1142.

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 plLac0-1 RBS Characterization Part – B0033
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