Team:Edinburgh UG/Parts

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.

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

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	http://parts.igem.org/Part:BBa_K2406020
Rox-Term-Rox	Test for function	BBa_K2406051	Terminator excised	http://parts.igem.org/Part:BBa_K2406051
Vox-Term-Vox	Test for function	BBa_K2406053	Terminator excised	http://parts.igem.org/Part:BBa_K2406053
Lox-Term-Lox*	Test for function	BBa_K2406055	Terminator excised	http://parts.igem.org/Part:BBa_K2406055
Vlox-Term-Lox	Orthogonality	BBa_K2406057	Terminator not excised	http://parts.igem.org/Part:BBa_K2406057
Vlox-Term-Vlox*	Test for function	BBa_K2406059	Terminator excised	http://parts.igem.org/Part:BBa_K2406059
Slox-Term-Lox*	Orthogonality	BBa_K2406061	Terminator not excised	http://parts.igem.org/Part:BBa_K2406061
Slox-Term-Vlox	Orthogonality	BBa_K2406063	Terminator not excised	http://parts.igem.org/Part:BBa_K2406063
Slox-Term-Slox/td>	Test for function	BBa_K2406065	Terminator excised	http://parts.igem.org/Part:BBa_K2406065
Vox-Term-Vlox	Orthogonality	BBa_K2406067	Terminator not excised	http://parts.igem.org/Part:BBa_K2406067
Vox-Term-Slox	Orthogonality	BBa_K2406069	Terminator not excised	http://parts.igem.org/Part:BBa_K2406069
Rox-Term-Slox	Orthogonality	BBa_K2406071	Terminator not excised	http://parts.igem.org/Part:BBa_K2406071
Rox-Term-Vox	Orthogonality	BBa_K2406073	Terminator not excised	http://parts.igem.org/Part:BBa_K2406073
Lox-Term-Vox	Orthogonality	BBa_K2406075	Terminator not excised	http://parts.igem.org/Part:BBa_K2406075
Vlox-Term-Rox	Orthogonality	BBa_K2406077	Terminator not excised	http://parts.igem.org/Part:BBa_K2406077
Lox-Term-Rox	Orthogonality	BBa_K2406079	Terminator not excised	http://parts.igem.org/Part:BBa_K2406079

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.

References

[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