Part:BBa_K3061002

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:

• 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 52
  Illegal NgoMIV site found at 61
  
• 1000
  COMPATIBLE WITH RFC[1000]