Team:TU Eindhoven/Basic Part
Basic Parts
Six basic parts have been developed. The basic parts are briefly described below.
dCas9 (BBa_K3168000) & dCas9-CP1041 (BBa_K3168001)
Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease which is part of the CRISPR-immune system of bacteria such as Streptococcus pyogenes. Both these basic parts encode the protein dCas9, which is a ‘dead’ variant of Cas9. dCas9 does not have its endonuclease activity, so it only binds to the target sequence and does not cut the DNA. The difference between these parts is that BBa_K3168001 is cysteine-free and a circularly permuted variant of dCas9 [1]. It has the same function and almost the same 3D structure, except for the location of the N- and C- terminus.
Large bit Nanoluc (BBa_K3168002) & Small bit NanoLuc (BBa_K3168003)
BBa_K3168002 and BBa_K3168003 encode for the large bit and the small bit of Split-NanoLuc respectively. When these two components find each other in the presence of the substrate (furimazine), blue light is emitted (Figure 1). These basic parts can be used to form fusion proteins that function as a split reporter to study for example protein-protein interactions [2]. TU Eindhoven 2019 used these parts to form a split reporter system which detects specific dsDNA sequences.
Figure 1: Split-NanoLuc principle.
Cysteine-free-NanoLuc (BBa_K3168006)
This basic part encodes for cysteine-free-NanoLuc. NanoLuc is a deep-sea shrimp-derived luciferase, which has increased stability, smaller size and a >150-fold increase in luminescence compared to other luciferases [3]. TU Eindhoven 2019 used this part to develop a BRET (Bioluminescent Resonance Energy Transfer) based sensor.
HisTag-MBP-TEVsite (BBa_K3168008)
This part can be very useful to solve protein expression and purification issues of complex proteins. It can be incorporated in front of the protein you want to express. Afterward, this part can be cleaved. This basic part includes a His tag, maltose-binding protein (MBP) and a TEV protease recognition site. The His tag can be used for protein purification with nickel affinity chromatography. MBP slows down the folding of newly synthesized proteins and it directs the protein to the periplasm. This protein is used to control protein folding of complex proteins with disulfide bonds. The TEV protease recognition site allows the cleavage of this part from a fusion protein after expression.
NanoLuc-mNeonGreen (BBa_K3168009)
This part encodes for a NanoLuc-mNeonGreen reporter. NanoLuc is a deep-sea shrimp-derived luciferase while mNeonGreen is a tetrameric fluorescent protein. The emission spectrum of NanoLuc and the excitation spectrum of mNeonGreen overlap, which enables bioluminescence resonance energy transfer (BRET). The result is that, when furimazine is added, the green emission from mNeonGreen is observed instead of the blue emission of NanoLuc, without further external illumination (Figure 2). The closer the donor and acceptor are, the more efficient the BRET is. Therefore, the C terminus of mNeonGreen overlaps with the N terminus of NanoLuc; no linker is used.
NanoLuc and NanoLuc-mNeonGreen both convert the same substrate and have a similar stability over time and in various measurement conditions. Therefore, NanoLuc-mNeonGreen enables ratiometric measurements where NanoLuc-mNeonGreen acts as a calibrator luciferase, which allows time and concentration independent measurements. This results in measurements that are more reliable than non-ratiometric measurements due to the independence of these conditions. The concept can be implemented easily when performing measurements with NanoLuc and wishing to do ratiometric calibration measurements. (For more details see our measurement page!)
Figure 2: Schematic representation of BRET between the donor NanoLuc and the acceptor mNeonGreen.
Basic 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_K3168006 | Basic | Cysteine-free-NanoLuc | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 567 | |
| BBa_K3168008 | Basic | HisTag-MBP-TEVsite | Eva Hanckmann, Claire Michielsen, Harm van der Veer | 1257 | |
| ❤ | BBa_K3168009 | Basic | NanoLuc-mNeonGreen | Simone Wouters | 1287 |
References
- Oakes, B. L., Fellmann, C., Rishi, H., Taylor, K. L., Ren, S. M., Nadler, D. C., ... & Savage, D. F. (2019). CRISPR-Cas9 circular permutants as programmable scaffolds for genome modification. Cell, 176(1-2), 254-267.
- 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.
- 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.
