Team:TU Eindhoven/Part Collection
Part collection
The unspecific use of antibiotics is one of the main causes of antimicrobial resistance [1]. To fight antimicrobial resistance, we want to enable the detection of specific bacteria in less than an hour, so the treatment with antibiotics can be customized from the beginning. Therefore, parts have been developed which enable the detection of specific dsDNA in low concentrations. This brings us to the overall theme of this part collection, to detect specific dsDNA in low concentrations.
The composite parts in this collection consist of fusion proteins of two basic parts (Figure 1). The first basic part recognizes a specific DNA sequence. There are two variants of this basic part; dCas9 (BBa_K3168000) and dCas9-CP1041 (BBa_K3168001). They both encode the dCas9 protein, however, the location of the termini differs. These parts are able to recognize a specific DNA sequence after incubation with specific gRNA which are complementary to the targeted DNA sequence. The specific gRNA sequences needed are determined using our dCas9 gRNA finder tool.
The recognition of a specific DNA sequence is visualized by the second basic part of the fusion protein. The binding to DNA is translated into a light signal. The luciferase NanoLuc, which emits blue light, is used. Two systems have been developed. The first system, paired dCas9-Split-NanoLuc is based on the split variant of NanoLuc. Light is emitted when two dCas9 proteins, one with the small bit and one with the large bit of Split-NanoLuc, bind in close proximity. The other system, single dCas9-BRET, is based on NanoLuc and the incorporation of a Cy3 dye. In this case, red light is emitted because of bioluminescent resonance energy transfer. This only occurs when the Cy3 dye and NanoLuc are in close proximity, due to DNA binding of dCas9. The composite parts are described in more detail on the composite parts page.
Figure 1: Schematic representation of the part collection.
Parts
| Favorite | Name | Type | Description | Designer | Length |
|---|---|---|---|---|---|
| BBa_K3168000 | Basic | dCas9 | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 4104 | |
| ❤ | BBa_K3168001 | Basic | dCas9-CP1041 | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 4164 |
| BBa_K3168002 | Basic | LargeBitNanoLuc | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 576 | |
| BBa_K3168003 | Basic | SmallBitNanoLuc | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 135 | |
| 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_K3168006 | Basic | Cysteine-free-NanoLuc | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 567 | |
| ❤ | BBa_K3168007 | Composite | dCas9-BRET | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 4731 |
References
- Cassini, A.; Högberg, L. D.; Plachouras, D.; Quattrocchi, A.; Hoxha, A.; Simonsen, G. S.; Colomb-Cotinat, M.; Kretzschmar, M. E.; Devleesschauwer, B.; Cecchini, M.; et al. Attributable Deaths and Disability-Adjusted Life-Years Caused by Infections with Antibiotic-Resistant Bacteria in the EU and the European Economic Area in 2015: A Population-Level Modelling Analysis. Lancet Infect. Dis. 2019, 19 (1), 56–66.
