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	<h3>Results and Discussion</h3><br>		<h3>Results and Discussion</h3><br>
	We measured the enzyme-catalyzed luminescence intensity of the protein which is split in new site and the old one. The results are shown in figure 1.		We measured the enzyme-catalyzed luminescence intensity of the protein which is split in new site and the old one. The results are shown in figure 1.
−	The relative luminescence intensity of st-1 and sc-1 is significantly stronger than st-2 and sc-2. It could be inferred that the two new split protein can combine easier and the split site predicted by our model is better than LargeBit(BBa_K1761005) and SmallBit(BBa_K1761006). <br>	+	The relative luminescence intensity of st-1 and sc-1 is significantly stronger than st-2 and sc-2. It could be inferred that the two new split protein can combine easier and the split site predicted by our model is better than LargeBit(
		+	<a href="https://parts.igem.org/Part:BBa_K1761005">BBa_K1761005</a>) and SmallBit(<a href="https://parts.igem.org/Part:BBa_K1761006">BBa_K1761006</a>). <br>
	<img  src="https://static.igem.org/mediawiki/parts/9/9c/T--DUT_China_B--BBa_K3061005.jpg">		<img  src="https://static.igem.org/mediawiki/parts/9/9c/T--DUT_China_B--BBa_K3061005.jpg">
	<p>Figure 1. Comparison of nanoLuc catalyzed coelenterazine luminescence at different split sites		<p>Figure 1. Comparison of nanoLuc catalyzed coelenterazine luminescence at different split sites
	Control group：2700 μl mixed crude enzyme +300 μl deionized water		Control group：2700 μl mixed crude enzyme +300 μl deionized water
	Relative luminous intensity：Crude enzyme luminescence /（Blank contrast luminescence×Total Protein of crude enzyme solution）</p>		Relative luminous intensity：Crude enzyme luminescence /（Blank contrast luminescence×Total Protein of crude enzyme solution）</p>
−	Abbreviation:	+	Abbreviation:<br><br>
	st-1: Fusion protein of new N terminal of Guassia luciferase and Spytag<br><br>		st-1: Fusion protein of new N terminal of Guassia luciferase and Spytag<br><br>
	st-2: Fusion protein of LargeBit luciferase and Spytag<br><br>		st-2: Fusion protein of LargeBit luciferase and Spytag<br><br>

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Latest revision as of 00:03, 22 October 2019

N-nanoluc, N-terminal part of split Guassia luciferase

It is the N-terminal part of the split Gaussia luciferase, which can recover its activity of blue light catalytic emission after combining with its C-terminal part. It can catalyze at the condition of oxygen and coelenterazine. It is an reporter to study protein-protein interactions.

Usage and Biology

This part is the N-terminus of Gaussia luciferase. It has an affinity towards N-terminus of Gaussia luciferase,so when in close proximity they will come together and will give a luminescence signal[1]. Their luminescence intensity is stronger than LargeBit(BBa_K1761005) and SmallBit(BBa_K1761006).

Chracterization

Protocols

Directly break st-1, st-2, sc-1, sc-2 engineered bacteria and centrifuge to collect the supernatant crude enzyme solution. After determining the protein concentration, it is used for luminescence detection. st-1 and sc-1 are good sites predicted by the model. The sites in st-2 and sc-2 have already existed in the registry. Mix 1350 μL two split protein solution and incubate for 10 min. Add 300 μL coelenterazine solution(5 μM) and then incubate for 10 min. Detect the emission light at 480 nm to evaluate the activity of these two split sites.

Results and Discussion

We measured the enzyme-catalyzed luminescence intensity of the protein which is split in new site and the old one. The results are shown in figure 1. The relative luminescence intensity of st-1 and sc-1 is significantly stronger than st-2 and sc-2. It could be inferred that the two new split protein can combine easier and the split site predicted by our model is better than LargeBit( BBa_K1761005) and SmallBit(BBa_K1761006).

Figure 1. Comparison of nanoLuc catalyzed coelenterazine luminescence at different split sites Control group：2700 μl mixed crude enzyme +300 μl deionized water Relative luminous intensity：Crude enzyme luminescence /（Blank contrast luminescence×Total Protein of crude enzyme solution）

Abbreviation:

st-1: Fusion protein of new N terminal of Guassia luciferase and Spytag

st-2: Fusion protein of LargeBit luciferase and Spytag

sc-1: Fusion protein of new C terminal of Guassia luciferase and SpyCatcher

sc-2: Fusion protein of SmallBit and SpyCatcher

References

[1]England C G , Ehlerding E B , Cai W . NanoLuc: A Small Luciferase is Brightening up the Field of Bioluminescence[J]. Bioconjugate Chemistry, 2016:acs.bioconjchem.6b00112.

Sequence and Features