Difference between revisions of "Part:BBa K4195040"

Latest revision as of 09:35, 13 October 2022

Nanoluc

Biology

NanoLuc
NanoLuc is a novel engineered luciferase enzyme that relies on the substrate furimazine to produce high intensity, glow-type luminescence. With high stability, small size and bright luminescence, it is an attractive luminescent reporter (1).

Fig. 1 The bioluminescent reaction catalyzed by NanoLuc® luciferase (1).

Usage and design

NanoLuc was selected for its good performance in enhancing the report signal. We add BBa_K3222000, BBa_K4195068, BBa_B0034 and BBa_K731721 to construct the expression system and obtained the composite BBa_K4195154, which are assembled on the expression vector pSB1C3 by standard assembly. The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Characterization

1. In Vivo Verification

1) Agarose Gel Electrophoresis

After transferring the plasmid into BL21(DE3), colony PCR was used to certify the plasmid was correct. The expected result was obtained (981bp).

Fig. 2 The result of colony PCR. Plasmid pSB1C3.

2) Bioluminescence measurement

Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of NanoLuc is observed by measuring the bioluminescence as time progressed using microplate reader.

Results are documented in related pages: BBa_K4195141, BBa_K4195142, BBa_K4195143, BBa_K4195144, BBa_K4195145, BBa_K4195149, BBa_K4195150, BBa_K4195151.

2. In Vitro Verification

Plasmid was put into the cell-free system for expression. The expression behavior of NanoLuc is observed by measuring the bioluminescence as time progressed using microplate reader.

Results are documented in related pages: BBa_K4195146, BBa_K4195147, BBa_K4195148.

3. Identification and protein Purification

In order to get NanoLuc and further purify it, we added a His-tag (6×His) to the C-terminal of it. The coding sequence of NanoLuc-his was cloned into the expression vector pET-28a(+) by Gibson assembly. Then we transformed the plasmid into E. coli BL21(DE3). The positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.

Fig. 3 DNA gel electrophoresis of the colony PCR products of NanoLuc-his_pET-28a(+).
After being cultivated and induced by IPTG, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of AmpC-his (Fig. 3), the target protein (19.9 kDa) can be observed at the position around 14 kDa on the purified protein lanes (FR).

Fig. 4 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples. Target bands (32.4 kDa) can be observed at the position around 35 kDa.

4. Paper-based bioluminescence assay

In order to determine the relationship between protein concentration and fluorescence, we use furimazine as substrate to perform a bioluminescence assay. The filter paper was cut into circular pieces, and the substrate was spotted onto them. After that, a short video was taken while we add purified NanoLuc to pieces. The result showed that a light change from brightness to darkness with the protein concentration ranged from high to low.(Fig. 4).

Fig. 5 The result of paper-based bioluminescence assay. Nano-Glo@ Luciferase Assay Substrate was added (3 μL per piece).

Reference

1. C. G. England, E. B. Ehlerding, W. Cai, NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence. Bioconjug Chem 27, 1175-1187 (2016).

Sequence and Features