Difference between revisions of "Part:BBa K3061005"
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<h1>Usage and Biology</h1><br> | <h1>Usage and Biology</h1><br> | ||
| − | 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> | + | 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(<a href="https://parts.igem.org/Part:BBa_K3061005">BBa_K1761005</a>) and SmallBit(<a href="https://parts.igem.org/Part:BBa_K1761005">BBa_K1761006</a>).<br><br> |
<h1>Chracterization</h1><br> | <h1>Chracterization</h1><br> | ||
<h3>Protocols</h3><br> | <h3>Protocols</h3><br> | ||
Revision as of 23:59, 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 Spytagst-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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]