Coding

Part:BBa_K2728003

Designed by: Zhenhao Yan   Group: iGEM18_BGIC-Global   (2018-10-09)
Revision as of 13:23, 16 October 2018 by Bluepumpkin (Talk | contribs) (→‎Experimental Characterization)


NanoLuc

Basic Description

NanoLuc (Nluc) luciferase is a small enzyme (19.1kDa) engineered for optimal performance as a luminescent reporter. The enzyme is about 100-fold brighter than either firefly (Photinus pyralis) or Renilla reniformis luciferase using a novel substrate, furimazine, to produce high intensity, glow-type luminescence. The luminescent reaction is ATP-independent and designed to suppress background luminescence for maximal assay sensitivity.

Sequence and Features

NanoLuc luciferase possesses a number of physical properties that make it an excellent reporter protein:

  • very small, monomeric enzyme (171 amino acids; 513bp)
  • high thermal stability (Tm = 60°C)
  • active over a broad pH range (pH 6–8)
  • no post-translational modifications or disulfide bonds
  • uniform distribution in cells
  • emission spectrum well suited for bioluminescence resonance energy transfer (BRET; λmax = 465nM)


Origin

Nanoluc luciferase is a commercial product from PROMEGA. It was engineered by directed evolution from a deep-sea shrimp (Oplophorus gracilirostris) luciferase.

Experimental Characterization

We used the kit provided by Promega to test its luminescence expresssion. We transferred the plasmid into E. coli BL21 strain, a common bacterial strain for protein expression. After overnight culture and adding substrate, we were able to witness the luminescence of Nanoluc in contrast of normal bacteria. T--BGIC-Global--nanoluc.jpg


Potential Application

The nanoluc luciferase can be used as a reporter of formaldehyde. We can calculate the formaldehyde concentration according to the strength of luminescence and the model of luminescence-formaldehyde concentration. What’s more, it can be used in the product so that customers can judge the existence of formaldehyde by observing the luminescence.

Parts Verification Before Submission

We verified our parts in the lab before submission. They are reliable! Please feel free to apply them onto your project.=)

T--BGIC-Global--partsub1.png

Fig 1: PCR (to get targeted genes)


T--BGIC-Global--partsub2.png

Fig 2: Restriction Digestion


T--BGIC-Global--partsub3.png

Fig 3: Ligation


T--BGIC-Global--partsub4.png

Fig 4: Colony PCR


T--BGIC-Global--partsub5.png

Fig 5: Gel Verification


References

Goyet, E., et al., Fast and high resolution single-cell BRET imaging. Sci Rep, 2016. 6: p. 28231.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 46
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None