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In order for Build-A-Cell to become a reality, we recognized a need for a more inclusive standard: one based on functionality to allow for easy reuse of biological components. To accomplish this, we have created an expression guide, funcitonally based architecture, and a compatibility check to allow for the application of the standard.
The Expression Guide
The expression guide aims to arouse awareness for a quantitative expression level and the feasible accuracy of current technologies and to guide the user to the functionally needed bin. Starting with examples of expression levels in E. coli the user can get an understanding which level might be useful for this module. In the next section, we calculated bins of expression levels for different examples to visualize precision of nature and technologies: simple replication of cells, statistical noise for low and high active regulation systems, precision of ribosome calculators and bi-cistronic designs with translational coupling. In our example calculation we used 99% probability to enable forward engineering. With this format of visualization the user can easily find the necessary bin for his module and potential collaboration is enabled by a common language. In the last step we present possible constructs the user can choose from to match the number of genes and desired quantity and variety of expression levels. More technical details on these constructs are presented in the following section.
Functionally Based Architecture
In order to give the user the possibility to realize single and multiple ORFs with small variability in expression levels we collected proven and designed new constructs for mono- and polycistrons with and without transcriptional and/or translational coupling. For polycistrons with low complexity of regulation units we designed a new construct called “Daisy Chain” inspired by natural construction of operons. We use -1 frameshifts of stop-start-codons for translational coupling and integrate the genes’ RBSs into the upstream gene by optimizing codons using simple calculation steps and the EMOPEC database. Proof of concept has been reached within this summer, further validation and optimization can be run using a crowd-based strategy. More information on the construct and its’ advantages and disadvantages in section “Design”.
Compatibility Check
The compatibility check helps the user to ensure freedom to share the designed modules with the community by analyzing 3rd party rights, prior research and biological compatibility with the host. We created a tutorial for the user to check the designed sequence against Lens Patent Database and to analyze found patents for potential issues. Template queries with tested keywords help to run searches on publication databases, e.g., webofscience, SCOPUS, etc. . The biological compatibility test is a protocol to test new modules on toxicity in the chosen host using multiple expression levels. See more details in corresponding sections.
With this new standard in hand, we come to the iGEM community to call the largest team of synthetic biologists to join together to bring this massive coordination to life!
Tell us about your project, describe what moves you and why this is something important for your team.
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References
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Inspiration
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