Difference between revisions of "Part:BBa K3061002"
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<partinfo>BBa_K3061002 short</partinfo> | <partinfo>BBa_K3061002 short</partinfo> | ||
| − | It is | + | It is signal peptide of Channelrhodopsin 2. |
| − | + | ||
<html> | <html> | ||
<head></head> | <head></head> | ||
<body> | <body> | ||
<h1>1. Usage</h1> | <h1>1. Usage</h1> | ||
| − | This part is the signal peptide of | + | This part is the signal peptide of Channelrhodopsin 2（ChR2）, which will locate the fusion protein to eyespot.<br><br> |
<h1>2. Biology</h1> | <h1>2. Biology</h1> | ||
| − | <p>Channelrhodopsin 2（ChR2） is light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. It is located in the plasma membrane[1]. ChR2 depolarize the plasma membrane in the eyespot region on a millisecond time scale after light excitation. We use the signal peptide of ChR2 to locate fusion protein phyB_C- | + | <p>Channelrhodopsin 2（ChR2） is light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. It is located in the plasma membrane[1]. ChR2 depolarize the plasma membrane in the eyespot region on a millisecond time scale after light excitation. We use the signal peptide of ChR2 to locate fusion protein phyB_C-rluc<a href="https://parts.igem.org/Part:BBa_K3061002">BBa_K3061010</a>around eyespot. The signal peptide correctly predicted by SignalP 4.1 server (predicts signal peptides) (Figure 1):<p> |
<img src="https://static.igem.org/mediawiki/parts/3/3d/T--DUT_China_B--BBa_K3061002.png"> | <img src="https://static.igem.org/mediawiki/parts/3/3d/T--DUT_China_B--BBa_K3061002.png"> | ||
<center><p >Figure 1. Results of SignalP 4.1 server</p></center> | <center><p >Figure 1. Results of SignalP 4.1 server</p></center> | ||
<h1>3. Characterization</h1> | <h1>3. Characterization</h1> | ||
| − | We fused signal peptide ChR2 with green fluorescent protein clover，see BBa_K3061008 | + | We fused signal peptide ChR2 with green fluorescent protein clover，see <a href="https://parts.igem.org/Part:BBa_K3061008">BBa_K3061008</a> ,and observe the fluorescence of fusion with signal peptide ChR2 by Olympus FV-1000 laser confocal microscope, see |
| + | <a href="https://static.igem.org/mediawiki/parts/0/00/T--DUT_China_B--protocols3.pdf">protocols 3 </a>.The reporter Clover demonstrated reliable expression and were clearly visualized with respective excitation 505 nm and emission 515 nm[2]. And the fluorescence is concentrated around the cell membrane, especially on the eyespot, which will help locate our fusion protein PhyB-C-hrluc to the eyespot.<br> | ||
| + | <img src="https://static.igem.org/mediawiki/parts/d/dc/T--DUT_China_B--SP-20.jpeg"> | ||
| + | <p>Figure 2. Detection of the intracellular fluorescence of Clover accumulated in the cytoplasm by confocal laser microscopy</p> | ||
| + | <br><br> | ||
<h1>4. References</h1> | <h1>4. References</h1> | ||
[1]Lauersen K J , Kruse O , Mussgnug J H . Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit[J]. Applied Microbiology and Biotechnology, 2015, 99(8):3491-3503.<br><br> | [1]Lauersen K J , Kruse O , Mussgnug J H . Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit[J]. Applied Microbiology and Biotechnology, 2015, 99(8):3491-3503.<br><br> | ||
Latest revision as of 00:21, 22 October 2019
Sp, signal peptide for Chlamydomonas reinhardtti
It is signal peptide of Channelrhodopsin 2.
1. Usage
This part is the signal peptide of Channelrhodopsin 2（ChR2）, which will locate the fusion protein to eyespot.2. Biology
Channelrhodopsin 2（ChR2） is light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. It is located in the plasma membrane[1]. ChR2 depolarize the plasma membrane in the eyespot region on a millisecond time scale after light excitation. We use the signal peptide of ChR2 to locate fusion protein phyB_C-rlucBBa_K3061010around eyespot. The signal peptide correctly predicted by SignalP 4.1 server (predicts signal peptides) (Figure 1):
Figure 1. Results of SignalP 4.1 server
3. Characterization
We fused signal peptide ChR2 with green fluorescent protein clover，see BBa_K3061008 ,and observe the fluorescence of fusion with signal peptide ChR2 by Olympus FV-1000 laser confocal microscope, see protocols 3 .The reporter Clover demonstrated reliable expression and were clearly visualized with respective excitation 505 nm and emission 515 nm[2]. And the fluorescence is concentrated around the cell membrane, especially on the eyespot, which will help locate our fusion protein PhyB-C-hrluc to the eyespot.
Figure 2. Detection of the intracellular fluorescence of Clover accumulated in the cytoplasm by confocal laser microscopy
4. References
[1]Lauersen K J , Kruse O , Mussgnug J H . Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit[J]. Applied Microbiology and Biotechnology, 2015, 99(8):3491-3503.[2]Ueki, Noriko. Dyneins || Dynein-mediated photobehavioral responses in, Chlamydomonas[M]. 2018.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 52
Illegal NgoMIV site found at 61 - 1000COMPATIBLE WITH RFC[1000]