Part:BBa_K1965014
nLuc:M13
Introduction
This part is a genetic fusion of the N‑terminal fragment of split firefly luciferase (nLuc, UniProtKB - P08659, aa 2‑491) and a calmodulin (CaM)-binding domain of the skeletal muscle myosin light chain kinase (M13, UniProtKB - Q9H1R3). This part in combination with BBa_K1965015 (CaM(E104Q):cLuc) comprises a sensitive calcium sensor for detecting calcium concentrations above physiological level.
NLuc is the N terminal fragment of split firefly luciferase while M13 is a domain of the skeletal muscle myosin light chain kinase responsible for calmodulin binding upon calcium influx and plays a key role in muscle contraction. In our calcium sensor, the conformational change that promotes binding of CaM to M13 peptide upon calcium ion binding, brings the two fragments of split firefly luciferase together and reconstitutes its activity 1A If you want to know more about split calcium sensor design please click here . We show that the CaM(E104Q):cLuc protein (BBa_K1965015) for formation of the split calcium sensor is expressed in the cytoplasm of HEK293T cells (Figure 1B). Co-expression of CaM(E104Q):cLuc (BBa_K1965015) and nLuc:M13 (BBa_K1965014) resulted in a 10-fold change in luciferase activity upon stimulation with 10 µM ionophore.
(A) Scheme of the split calcium sensor. Calmodulin is linked to the C-terminal fragment and M13 to the N-terminal fragment of split firefly luciferase. An elevated intracellular concentration of calcium ions triggers binding of M13 to calmodulin and formation of active luciferase. (B) CaM(E104Q):cLuc was expressed in the cytosol. HEK293T cells were transfected with 10 ng of the CaM(E104Q):cLuc plasmid. 24 h after transfection cells were fixed with paraformaldehyde and permeabilized, stained with anti-HA antibodies. Localization was confirmed with confocal microscopy. (C) Fold activation of the split calcium sensor was dependent of the ratio between the CaM(E104Q):cLuc and the nLuc:M13 plasmids. HEK293T cells were transfected with plasmids, encoding the split calcium sensor (CaM(E104Q):cLuc and nLuc:M13). 24 h after transfection luciferase activity was measured immediately after the addition of 10µM ionophore A23187.
The split calcium sensor is compatible with different constructs and different means of stimulation. In addition to ionophore stimulation, it also responds to stimulation by direct contact that underlies the sense of touch, where calcium enters cells via stimulation of a designed mechano-responsive system. In addition to the split calcium sensor, HEK293 cells were transfected with the following constructs: BBa_K1965000 (coding for a bacterial mechanosensitive receptor MscS), BBa_K1965003 and BBa_K1965004 (coding for proteins GvpC and GvpA, respectively, enabling formation of gas vesicles) ( 2A . As shown in Figure 2B, the cells were able to convert mechanical stimulus (in this case, touching with a glass rod ( touchpaint ) into light signal by activation of the calcium sensor and reconstitution of split luciferase.
(A) Schematic presentation of a cell with increased sensitivity to mechanical stimulation due to expression of a mechanosensitive ion channel MscS and gas vesicle-forming proteins. Upon mechanical stimulation, calcium enters the cell via MscS channels, enabling activation of the calcium sensor and reconstitution of the split luciferaseB) Images of petri dishes seeded with HEK293 cells, expressing the mechano-responsive calcium sensing system after mechanical stimulation .24h after transfection, luciferin and CaCl2 were added to the media and the cells were stimulated by touching with a glass rod. Afterwards, camera images were taken in darkness with exposure time 30 s.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 103
Illegal NgoMIV site found at 1447
Illegal NgoMIV site found at 1468
Illegal AgeI site found at 1171 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1353
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