Difference between revisions of "Part:BBa K3061005"
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<h1>Usage</h1><br>
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<h1>Usage and Biology</h1><br>
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This part is the N-terminus of intact Gaussian Renilla luciferase, and the split Gaussian kidney luciferase is often used for interaction protein detection. We link the split Renilla luciferase to the interaction protein BBa_K1159200, BBa_K1159201 in the registry. When the interaction protein binds, Renilla Luciferase also restores its activity, thereby understanding its activity by detecting the luminescence intensity of its catalytic substrate.<br>
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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).<br><br>
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Contrast with the old part:<br>
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Initially, we used split Nanoluc from BBa_K1761005, an interaction protein from BBa_K1159200. After testing, we found that the split Nanoluc has very low binding efficiency and hardly catalyzes substrate luminescence. After the model detection, we found that the cleavage site in the part BBa_K1761005 is not effective. We designed a new cleavage site by designing the scoring function. According to the experimental results, the cleavage site has a better effect.<br>
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<h1>biology</h1><br>
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Split Renilla luciferase has outstanding applications in protein interaction detection, biomedicine, and cell development, but it also has the disadvantages of low catalytic efficiency and high background value. In our previous experiments, the complete catalytic activity of Renilla luciferase was poor, and when directly expressed in algae or E. coli, membrane-transparent coelenterazine was directly added to the above-mentioned engineering microorganisms, and the luminescence was not detected. Therefore, we are eager to find a more active enzyme - Nanoluc.NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future[1].<br>
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<h1>Chracterization</h1><br>
 
<h1>Chracterization</h1><br>
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<p>1 protocols<br></p><br>
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<h3>Protocols</h3><br>
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st-1: the combination of new N terminal of Guassia luciferase and Spytag  <br>
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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.<br><br>
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st-2: the combination of N terminal of Guassia luciferase and Spytag  <br>
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<h3>Results and Discussion</h3><br>
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sc-1: the combination of new C terminal of Guassia luciferase and SpyCatcher <br>
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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.  
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sc-2: the combination of C terminal of Guassia luciferase and SpyCatcher  <br>
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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>
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Directly break st-1, st-2, sc-1, sc-2 engineered bacteria induced and collect supernatant crude enzyme solution by centrifugation. 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. Add 1350 μl each of these two interacting crude enzyme solutions in 3 ml system. Incubate for 10min and wait for the combination of Spytag and SpyCatcher, in order to restore the activity of NanoLuc. Add 300 μl coelenterazine solution and incubate it for 10 min to detect the emission light at 480 nm, so as to determine the activity of these two splitting sites.<br>
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<p>2 Result and Discussion</p><br><br>
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According to the above experimental scheme, we measured the enzyme-catalyzed luminescence intensity of the different split sites. The results are shown in figure 1. The relative luminescence intensity of st-1 and sc-1 is significantly different from which of st-2 and sc-2. It could be inferred that the split site predicted by the model is better. Our subsequent experiments can be guided by the model to cut luciferase more precisely.<br>
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<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>
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Abbreviation:
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<h1>4.References</h1>
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st-1: Fusion protein of new N terminal of Guassia luciferase and Spytag<br><br>
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st-2: Fusion protein of LargeBit luciferase and Spytag<br><br>
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sc-1: Fusion protein of new C terminal of Guassia luciferase and SpyCatcher<br><br>
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sc-2: Fusion protein of SmallBit and SpyCatcher<br><br>
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<h1>References</h1>
 
[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.
 
[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.
  

Revision as of 23:48, 21 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]