In order to determine the appropriate expression levels of various component genes, our project required us to create and test many circuit variants. To efficiently design and clone circuits containing multiple transcriptional units, we implemented a recently designed method of circuit construction called 3G Assembly, which enables quick and modular cloning of circuits. Furthermore, mixed” 3G Assembly can be used to construct and test multiple variants of a given circuit. This allows teams to build, test and subsequently clone a vast number of circuit variants in a single day.

Since we anticipate this method to be extremely valuable to future iGEM teams, we submitted approximately 70 parts in a 3G compatible format. These parts include some of the most commonly used basic parts used by iGEM teams, as well as basic parts from our project and parts from our collaborators at UVA.

Our 3G part library consists of 4 categories: Promoter, 5’ UTR (UnTranslated Regions), Coding sequence and Terminator. Most of the variants in each category are used for tuning the relative expression level between the reporter (mScarlet) and the protease (mf-Lon). We can also easily modify degradation rate by switching the degradation tag in mScarlet, changing the strength of the promoter regulating mf-Lon, or adding a degradation tag to mf-Lon (a ssrA tag).

In our collection of parts, we also incorporated the ts-CI heat inducible system and TlpA heat inducible system. To use these systems, we cloned the corresponding promoter and repressor sequences needed into our standard backbone. Using these heat inducible parts, we also designed a wide variety of heat inducible circuits. Their circuits are a convenient tool for any team in the future that are using 3G methods and are pursuing this induction system.

While our part collection is complete for the purpose of our project, it is also open to addition. A 3G library will be an invaluable tool for other teams also hoping to implement 3G assembly, and thus the library will be open-access for all iGEM teams to contribute to.