Part:BBa_K4195154

T7-Nanoluc-T7t

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.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 619
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 38
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 141
  
• 1000
  COMPATIBLE WITH RFC[1000]