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<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >This year our team has been focusing on creating a system that will allow other iGEM teams to easily control the gene expression speed of an arbitrary protein without requiring a costly wholesale redesign of existing architecture. To that end we characterized how protein degradation tags (pdt) can be used to modularly change the speed of mScarlet-I, a fast folding (7 minutes) high quantum yield red fluorescent protein. We also created a set of easy cloning parts which enable future iGEM teams to clone our parts without needing intermediates. At the UVA Mid-Atlantic meetup, we were particularly intrigued by University of Maryland iGEM’s copper sensor. After discussing with them, we agreed that we would collaborate on improving the function of their copper sensor (BBa_ BBa_K2477013). Since they primarily wanted to use their copper sensor as an educational tool, reducing the time required to get results would help them keep students engaged, as well as enable them to use their device in labs or outreach activities that did not have a lot of time. </div>
One of the main goals of synthetic biology is to create a modular genetic basis for the independent control of circuit behavior properties. Much progress has been made in achieving this aim for properties like gene expression strength (where well-characterized ribosome binding sites (RBSs) can be swapped within a genetic part), circuit architecture (where promoters can be swapped out to introduce connections and feedback architectures), and even gene expression noise (through a combination of the above two modulations). However, in order to move into the next phase of synthetic biology, we need to be able to control the dynamical properties of circuits— we want to move beyond circuits that focus on endpoint, steady-state values and explore the rich variety of dynamical systems. Fundamentally, gaining controlling of dynamical systems implies gaining control of temporal dynamics.
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<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >To help them increase their gene expression speed we proposed that we jointly create and characterize modified versions of their parts with pdts. Since time was short and Maryland did not have access to a cell sorter, we simply had them ship us their parts, and then did the modifications and characterizations at William and Mary, before sending the modified parts back for use. On the other end, William and Mary sent blinded speed constructs to Maryland for testing. We hoped that this would enable us to confirm degradation in platforms other than our own, and potentially show speed change as well.</div>
Currently, there is no good way to control the temporal dynamics of gene expression. Current control strategies require either a rewiring of the circuit architecture to achieve different time-dependent dynamics [1, 2] or a complete circumvention of transcriptional circuitry altogether, relying on post-translational dynamics like phosphorylation [3] or protein-protein interactions [4] to transmit information through a circuit. These approaches are often inaccessible to iGEM teams because they require too drastic an overhaul of existing circuit implementations. To alleviate this issue, and to enable future iGEM teams to create robust dynamical circuits, we created a protein degradation based ‘plug-and-play’ style system that allows modular and predictable control of the gene expression speed of a given circuit without requiring a fundamental redesign of existing circuit architecture.
<figcaption><div style='padding-left: 0px;'>Figure 1: Schematic of University of Maryland's modified copper sensing part </div></figcaption>
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Revision as of 20:37, 30 October 2017
Motivation
One of the main goals of synthetic biology is to create a modular genetic basis for the independent control of circuit behavior properties. Much progress has been made in achieving this aim for properties like gene expression strength (where well-characterized ribosome binding sites (RBSs) can be swapped within a genetic part), circuit architecture (where promoters can be swapped out to introduce connections and feedback architectures), and even gene expression noise (through a combination of the above two modulations). However, in order to move into the next phase of synthetic biology, we need to be able to control the dynamical properties of circuits— we want to move beyond circuits that focus on endpoint, steady-state values and explore the rich variety of dynamical systems. Fundamentally, gaining controlling of dynamical systems implies gaining control of temporal dynamics.
Currently, there is no good way to control the temporal dynamics of gene expression. Current control strategies require either a rewiring of the circuit architecture to achieve different time-dependent dynamics [1, 2] or a complete circumvention of transcriptional circuitry altogether, relying on post-translational dynamics like phosphorylation [3] or protein-protein interactions [4] to transmit information through a circuit. These approaches are often inaccessible to iGEM teams because they require too drastic an overhaul of existing circuit implementations. To alleviate this issue, and to enable future iGEM teams to create robust dynamical circuits, we created a protein degradation based ‘plug-and-play’ style system that allows modular and predictable control of the gene expression speed of a given circuit without requiring a fundamental redesign of existing circuit architecture.
Results
We managed to clone and sequence all 6 pdt variants of University of Maryland’s copper circuit (K2333437-K2333442). We confirmed that they are inducible with copper sulfate and that the RFP produced can be degraded by mf-Lon (Figure 1). We then performed preliminary speed characterization of a subset of the parts before sending them back to Maryland (Figure 2). We also analyzed Maryland’s data and found that degradation was in fact occurring (Figure 3). Together these results show that not only can our system degrade in different strains and media conditions, but that it can degrade and change the speed of gene expression for arbitrary proteins. While we didn’t get to characterize the modified parts to the extent we wanted, we did see a noticeable and qualitatively similar speed change in our characterization. We hope that with a little more testing, we’ll be able to make these parts as consistent as our test parts.
We would also like to note that due to a judging form mishap, we did not fill in the Silver Medal parts requirement with any BioBrick IDs. However, since these parts serve as both new parts and functional proofs of concept, they would be sufficient to fulfill the Silver Medal requirement, we would humbly ask that judges evaluate these parts (among others), as proof of our fulfillment of Silver Medal requirement I. More information on this can be found on our for Judges pages, as well as our other part related pages.
