Parts

Introduction

The main goal of our project in the short-term was to develop and test recombinase parts in E. coli. To the best of our knowledge, these parts have not been extensively tested for activity and orthogonality in bacterial cells. The parts listed below have all been functionally verified, and can be used to construct the systems described on our design page [link]. We report verified function in four tyrosine recombinases, none of which have been correctly documented by any iGEM team before. We also report target sites for each recombinase, none of which have been properly documented or entered into the registry before. We have also developed a collection of orthogonal Cre-recognised Lox sites. Finally, we have developed measurement constructs for each recombinase. We used these constructs to measure and confirm the activity of our basic parts.

Basic parts

Our basic parts fall into three categories: Recombinase generators, recombinase target sites, and orthogonal target sites for Cre. We selected the recombinases Dre [1], Vika [2], VCre, and SCre [3] to form the basis of our toolkit. These recombinases were shown to not cross-react with the Cre/LoxP system [1][2][3][5], and, in the case of VCre and SCre [3], were claimed to not cross-react with one another. As part of our toolkit, we also included the associated target sites for these recombinase generators. For a snapshot of how we verified the function of these parts, scroll down to the “Demonstrate” section of this page. For a more detailed view, click on the parts registry link, which will take you to the detailed analysis of each individual part. We also included in the toolkit orthogonal target sites for Cre recombinase. Cre/LoxP is a very popular recombinase system that is frequently used in biotechnology [4]. In order to expand the potential of this system, we have developed multiple mutant Lox target sites, which have previously been identified as sites that will not react with the classic LoxP site [4]. Adding these mutant, orthogonal lox sites to the toolkits means we provide a framework whereby one Cre protein could catalyse up to ten distinct recombination events within one cell. Once again, check out the full registry pages for each part to learn more and download their individual sequences. To simplify our experimental workflows, we have also developed an inducible Cre recombinase generator, also reported below.

Composite parts

Our generators use our novel T7-LacO promoter composite. This allows for inducible expression of our parts in E. coli strains expressing T7 Polymerase. As is elaborated on the improve section of this page, this constitutes a valuable part improvement. Finally, we assembled measurement constructs. These consisted of two target sites flanking a promoter, with this part inserted in between the promoter and RBS of the BBa_J04450 part. As explained in the diagram below, these constructs could test if two target sites would recombine when a particular recombinase was present in the cell. This allowed us to demonstrate our recombinase target sites, recombinase coding sequences, and novel promoter functioned as anticipated. Furthermore, they allowed for us to demonstrate which target sites for different recombinases would not recombine with one another. It should be noted we did observe some unexpected cross-reactivity. In a sense, this is a positive, as it verifies the validity of our measurement construct for testing if two recombinase sites are orthogonal. Indeed, it appears our measurement constructs are more sensitive than those reported in the literature, as cross reactivity for SCre and VCre had not previously been observed [3].

Note: * indicates parts that were not ultimately constructed due to time constraints.