Team:Edinburgh UG/Parts



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.

Part Part Type Registry Name Functional Verification Registry Link
T7-LacO-Dre Basic Recombinase generator BBa_K2406081 Shown to excise terminator inducibly
T7-LacO-Vika Basic Recombinase generator BBa_K2406082 Shown to excise terminator inducibly
T7-LacO-Vcre Basic Recombinase generator BBa_K2406083 Shown to excise terminator inducibly
T7-LacO-Scre Basic Recombinase generator BBa_K2406084 Shown to excise terminator inducibly
T7-LacO-Dre Basic Recombinase generator BBa_K2406081 Shown to excise terminator inducibly
Rox Recombinase target site BBa_K2406000 Excise terminator, orthogonal test
Vox Recombinase target site BBa_K2406001 Excise terminator, orthogonal test
Vlox Recombinase target site BBa_K2406002 Excise terminator, orthogonal test
Slox Recombinase target site BBa_K2406003 Excise terminator, orthogonal test
Lox511 Orthogonal Lox site BBa_K2406008 Sequencing, functional assay
Lox2272 Orthogonal Lox site BBa_K2406009 Sequencing, functional assay
Lox5171 Orthogonal Lox site BBa_K2406010 Sequencing, functional assay
LoxN Orthogonal Lox site BBa_K2406011 Sequencing, functional assay
M2 Orthogonal Lox site BBa_K2406012 Sequencing, functional assay
M3 Orthogonal Lox site BBa_K2406013 Sequencing, functional assay
M7 Orthogonal Lox site BBa_K2406014 Sequencing, functional assay
M11 Orthogonal Lox site BBa_K2406015 Sequencing, functional assay
Nuoya Orthogonal Lox site BBa_K2406016 Sequencing, functional assay
Zsoka Orthogonal Lox site BBa_K2406017 Sequencing, functional assay
T7-LacO-Cre Recombinase generator BBa_K2406080 Sequencing, functional assay

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].

Fig. 1: Basic schematic demonstrating principle of our recombinase measurement constructs. Parallel lines (or capacitor for engineers) represents transcription terminator downstream of a promoter. Triangles of identical colours represent target sites that can recombine with one another. Different coloured triangles are incompatible target sites. Note that RFP reporter gives off quantitative output, so recombination efficiency between two sites can also be quantified.

Part Part Type Registry Name Functional Verification Registry Link
T7-LacO Promoter BBa_K2406020 Sequencing, inducible production
Rox-Term-Rox Test for function BBa_K2406051 Terminator excised
Vox-Term-Vox Test for function BBa_K2406053 Terminator excised
Lox-Term-Lox* Test for function BBa_K2406055 Terminator excised
Vlox-Term-Lox Orthogonality BBa_K2406057 Terminator not excised
Vlox-Term-Vlox* Test for function BBa_K2406059 Terminator excised
Slox-Term-Lox* Orthogonality BBa_K2406061 Terminator not excised
Slox-Term-Vlox Orthogonality BBa_K2406063 Terminator not excised
Slox-Term-Slox/td> Test for function BBa_K2406065 Terminator excised
Vox-Term-Vlox Orthogonality BBa_K2406067 Terminator not excised
Vox-Term-Slox Orthogonality BBa_K2406069 Terminator not excised
Rox-Term-Slox Orthogonality BBa_K2406071 Terminator not excised
Rox-Term-Vox Orthogonality BBa_K2406073 Terminator not excised
Lox-Term-Vox Orthogonality BBa_K2406075 Terminator not excised
Vlox-Term-Rox Orthogonality BBa_K2406077 Terminator not excised
Lox-Term-Rox Orthogonality BBa_K2406079 Terminator not excised

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

Silver Medal Criteria: New Parts

Our team most straightforwardly satisfies this requirement with our recombinase generators for Dre, Vika, Vlox, and SCre. Other teams have claimed to have developed generators before. However, none with the sequences we have used have been submitted and none have been documented at all. Therefore, these recombinase coding sequences should be recognised as new parts: there are no parts with the design, sequences, or function of our new coding sequence parts. As demonstrated by the data on each parts registry page, each has been sequence confirmed and demonstrated to function via our measurement constructs. A “snapshot” of the data you can find on the registry page is shown below. Below are the results of the assays involving Dre recombinase. As demonstrated by the Rox-Term-Rox measurement construct, Dre recognises and causes recombination between Rox sites, as very high RFP fluorescence was observed. Conversely, negligible fluorescence was observed in all other measurement constructs, demonstrating the orthogonal nature of the Dre/Rox system.

Gold Medal Criteria: Improve a Previous Part

Our team has created functional variants of the basic lox part and vastly improved the documentation of the individual recombinase target sites (Rox, Vox, Vlox, and Slox). This all counts as part improvement. The simplest, easiest to demonstrate improvement is our new T7-LacO promoter, though. Previous attempts have been made to create this inducible T7 promoter. However, none have been previously documented to work on the registry. Furthermore, our part has subtle sequence differences to previous parts, although it is unclear if this is the reason for the function of our promoter, as there is no data regarding previous attempts to make this part. Therefore, our part represents an improvement on parts BBa_R0184, BBa_R0185, BBa_R0186, and BBa_R0187. Documentation on the parts registry page demonstrates the function of our promoter. The utility of the part is demonstrated by its ability to decrease leakiness through a two-tiered repression system, illustrated below.

Fig. 2: T7 Polymerase is under control of standard Lac promoter. The Gene of interest is under the control of T7 Polymerase promoter that is repressed by LacI. Therefore, incubation with IPTG will cause T7 polymerase to be produced and repression to be removed from the gene of interest’s promoter. This is less leaky than a standard induction system. That is because, for there to be leaky production of the gene of interest, first, there must be a “leak” at the Lac Promoter. Then, there must be a subsequent “leak” at the T7-LacO promoter. Therefore, this is less likely to occur than a solitary “leak” from a normal LacO-based promoter.

Part collection

Our part collection consists of all of the basic and composite parts listed above. Our team is very proud of the number of parts we have successfully assembled and functionally verified. For this reason, and the reasons outlined below, we feel we qualify for the Best Part Collection special prize. Our parts are all well-characterised, verified to work, and have all of this information strongly documented on the iGEM registry. These parts form the backbone of our toolkit, and are intended to be used together, as the exciting possibilities of using multiple orthogonal recombinases in concert is what inspired us to choose to develop our toolkit and part collection in the first place. Our parts are all ready to use, and can readily be assembled into other, new, exciting constructs simply by using parts on the registry. Our team has demonstrated the capacity to build more complex systems using these parts, demonstrated best by our measurement constructs. Each measurement construct required two recombinase target sites to be integrated into the BBa_J04450 and for the cells to also be transformed with a recombinase generator. Therefore, we demonstrated that our parts collection can be used to assemble higher-order constructs. Finally, the documentation of our parts is complete with information on each recombinase’s registry page that denotes which other parts it is suitable to use with. Therefore, other teams will be able to take advantage of our parts and documentation to develop their own unique projects.


[1] Anastassiadis, K., Fu, J., Patsch, C., Hu, S., Weidlich, S., Duerschke, K., Buchholz, F., Edenhofer, F., and Stewart A.F. 2009. “Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice.” Disease Models and Mechanisms: Sep-Oct; 2(9-10):508-515.

[2] Karimova, M., Abi-Ghanem, J., Berger, N., Surendranath, V., Pisabarro, M.T., Buchholz, F. 2013 “Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system”. Nucleic Acids Research 41(2):e37.

[3] Suzuki E. and Nakayama, M. 2011. “VCre/VloxP and SCre/SloxP: new site-specific recombination systems for genome engineering.” Nucleic Acids Research 39(8):e49

[4] Missirlis, P.I., Smailus, D.E., and Holt, R.A. 2006. “A high-throughput screen identifying sequence and promiscuity characteristics of the loxP spacer region in Cre-mediated recombination”. BMC Genomics 7: 73. s

[5] Liu, W., Tuck, L.R., Wright, J.M, and Cai, Y. Using Purified Tyrosine Site-Specific Recombinases In Vitro to Rapidly Construct and Diversify Metabolic Pathways. 2017. Methods Mol Biol. 1642: 285-302