Difference between revisions of "Part:BBa K2728003"

Latest revision as of 03:09, 17 October 2018

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.

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.=)

Fig 1: PCR (to get targeted genes)

Fig 2: Restriction Digestion

Fig 3: Ligation

Fig 4: Colony PCR

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]

@@ Line 24: / Line 24: @@
 === Experimental Characterization ===
-We used the original plasmid provided by Promega to verify its luminescence strength. We transferred the plasmid into E. coli BL21 strain, a common bacterial strain for protein expression. 200 μl of the overnight bacteria was added into the 96-well plate for the luminescence assay. The luminescence curve was tested under the condition of emission wavelength=465nM and orbital shaking with amplitude=2. We can observed from the result that
+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.
 <br />
+[[File:Ep5.png|left|500px]]<br clear=all>
+[[File:T--BGIC-Global--nanoluccorrection.jpg|left|500px]]<br clear=all>
 <br />
+<br />
 === 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.