Team:TU Eindhoven/Composite Part
Composite parts
The aim of our composite parts is to detect specific dsDNA in low concentrations. dCas9 is used to target unique DNA sequences. Guide-RNA (gRNA) enables dCas9 to target a specific sequence. The unique gRNA sequences needed are determined using our dCas9 gRNA finder tool. Two systems were developed to create a signal when dCas9 binds to its target sequence.
Paired dCas9-Split-NanoLuc system
The first method is based on the Peking 2015 team. Two dCas9 proteins bind in close proximity of each other, which enables the attached split luciferases to form a complex and produce light. Peking 2015 used the firefly luciferase, which is a relatively large and unstable luciferase. We improved this part by incorporating NanoLuc, a 19kDa luciferase which is a thousand-fold brighter and has increased stability [1]. The split version of NanoLuc consists of a large bit and a small bit. This paired dCas9-Split-NanoLuc system consists of two composite parts, which are described below.
dCas9-SmallBitNanoLuc (BBa_K3168005)
This composite part encodes for a fusion protein of dCas9 (BBa_K3168000) and NanoLuc small bit (BBa_K3168005) with an affinity of 2.5*10-6M. NanoLuc small bit and dCas9 are fused using a (GGS)5 linker. The affinity of the small bit can be varied by incorporating a few specific mutations [2]. A low affinity was chosen, so the NanoLuc complex is only formed when the two dCas9 proteins bring the two components in close proximity through binding to their target sequence (Figure 1).
dCas9-LargeBitNanoLuc (BBa_K3168004)
This composite part encodes for a fusion protein of dCas9 (BBa_K3168000) and NanoLuc large bit (BBa_K3168004), which also contains a (GGS)5 linker between the two components. A schematic representation of the two composite parts interacting with each other is shown in Figure 1.
Figure 1: Schematic representation of the dCas9-Split-NanoLuc system.
Single dCas9-BRET system
The second method uses Bioluminescent Resonance Energy Transfer (BRET) to create a ratiometric signal, which decreases the background signal. BRET relies on a resonance energy transfer from a bioluminescent donor to an acceptor in the presence of the substrate (furimazine). The donor and acceptor need to be in close proximity and the emission spectrum of the donor needs to overlap with the excitation spectrum of the acceptor.
The single dCas9-BRET system is based on the conformational change of dCas9 when binding DNA [3]. A Cy3 dye is coupled to dCas9, which is fused to NanoLuc via a (GGS)2 linker. When the fusion protein binds DNA, the conformational change brings the Cy3 dye in close proximity with NanoLuc, which enables BRET (Figure 2). No BRET is present when dCas9 is not bound to the target DNA, as the dye and NanoLuc are too far away from each other.
Figure 2: Schematic representation of the dCas9-BRET system.
dCas9-BRET (BBa_K3168007)
The single dCas9-BRET system consists of one composite part, which is a fusion protein of dCas9 (BBa_K3168001) and cysteine free NanoLuc (BBa_K3168006) with a (GGS)2 linker between the components. When a Cy3 dye is incorporated, the conformational change of the HNH domain when dCas9 binds to DNA can be detected [3]. This Cy3 dye needs to be incorporated in the HNH domain via maleimide coupling. Therefore, the original cysteines in dCas9 and NanoLuc were mutated and one cysteine was incorporated in the HNH domain for the coupling of the dye (S187C) after protein expression.
Composite parts
| Favorite | Name | Type | Description | Designer | Length |
|---|---|---|---|---|---|
| ❤ | BBa_K3168004 | Composite | dCas9-LargeBitNanoLuc | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 4680 |
| ❤ | BBa_K3168005 | Composite | dCas9-SmallBitNanoLuc | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 4239 |
| BBa_K3168007 | Composite | dCas9-BRET | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 4731 |
References
- England, C. G., Ehlerding, E. B., & Cai, W. (2016). NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjugate chemistry, 27(5), 1175-1187.
- Dixon, A. S., Schwinn, M. K., Hall, M. P., Zimmerman, K., Otto, P., Lubben, T. H., ... & Wood, M. G. (2015). NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS chemical biology, 11(2), 400-408.
- Sternberg, S. H., LaFrance, B., Kaplan, M., & Doudna, J. A. (2015). Conformational control of DNA target cleavage by CRISPR–Cas9. Nature, 527(7576), 110.
