<div style = 'padding-left: 190px; padding-bottom: 10px;font-size: 25px' ><b>Degradation Based Control of Gene Expression Speed</b></div>
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<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >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.</div>
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<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >Preliminary mathematical analysis reveals that degradation rate is the key factor in controlling gene expression speed. Consider the simple kinetic model....</div>
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<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >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.</div>
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<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >Thus in order to create a plug-and-play style system to control gene expression speed, we developed and characterized a suite of BioBrick parts which allow for simple, modular and predictable changes to the gene expression speed of arbitrary proteins.</div>
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<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >When we were created our gene expression control system, we wanted to make sure that it was both usable across a variety of biological systems and circuits, and easily accessible to other iGEM teams. To this end, we chose to use the Mesoplasma florum Lon (mf-Lon) protease system discovered by Gur and Sauer in 2008, and developed by Collins and Cameron in 2014. This system consists of of a AAA+ protease and its associated protein degradation tags (pdt), which operate in a mechanistically similar manner to the E. Coli endogenous protease ClpXP and its associated ssrA tags. However, unlike ClpXP and ssrA tags, mf-Lon and pdts are completely orthogonal to the endogenous protein degradation systems in E. coli. Using this orthogonal degradation system helps eliminate cross talk between our system and endogenous E. coli proteins. Further, since there are pdts with a wide range of different affinities, we are able to tune degradation rate, and thus gene expression speed to a wide variety of values. This represents not only a practical advancement in the tuning of circuits, but also serves as the first report of the previously unconfirmed mathematical prediction of the relationship between degradation rate and gene expression speed.</div>
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<div style = 'padding-right: 190px; padding-left: 190px; text-indent: 50px;line-height: 25px;' >Math stuff. Plus we need a click through to results</div>
Revision as of 20:42, 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.
Degradation Based Control of Gene Expression Speed
Preliminary mathematical analysis reveals that degradation rate is the key factor in controlling gene expression speed. Consider the simple kinetic model....
Thus in order to create a plug-and-play style system to control gene expression speed, we developed and characterized a suite of BioBrick parts which allow for simple, modular and predictable changes to the gene expression speed of arbitrary proteins.
Orthogonal Degradation Tags
When we were created our gene expression control system, we wanted to make sure that it was both usable across a variety of biological systems and circuits, and easily accessible to other iGEM teams. To this end, we chose to use the Mesoplasma florum Lon (mf-Lon) protease system discovered by Gur and Sauer in 2008, and developed by Collins and Cameron in 2014. This system consists of of a AAA+ protease and its associated protein degradation tags (pdt), which operate in a mechanistically similar manner to the E. Coli endogenous protease ClpXP and its associated ssrA tags. However, unlike ClpXP and ssrA tags, mf-Lon and pdts are completely orthogonal to the endogenous protein degradation systems in E. coli. Using this orthogonal degradation system helps eliminate cross talk between our system and endogenous E. coli proteins. Further, since there are pdts with a wide range of different affinities, we are able to tune degradation rate, and thus gene expression speed to a wide variety of values. This represents not only a practical advancement in the tuning of circuits, but also serves as the first report of the previously unconfirmed mathematical prediction of the relationship between degradation rate and gene expression speed.
Modeling
Math stuff. Plus we need a click through to results
