Part:BBa_K2271021

pHlourin2 - pH sensitive green flourescent protein for yeast expression

Basic part. Only gene sequence.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 10
  

The activity of enzymatic Proteins is mostly pH-dependent. Therefore, it is of high interest to understand the pH-regulating mechanism of the peroxisome and the effects on the imported pathways. Literature has not agreed whether there is a common peroxisomal pH nor whether there is a regulating mechanism (Francesco M. Lasorsa et al.(2004)and Carlo W. T. van Roermundet al. (2004) . For our measurements, we use pH Lourin2, a GFP variant with a bimodal excitation spectrum with peaks at 395 and 475 nm and an emission maximum at 509 nm. Upon acidification, the excitation spectrum shifts from 395 to 475 nm.
We cloned pHlourin2 part into a cassette with n-terminal 3X-Flag, with and without c-terminal peroxisomal targeting sequence 1. GFP filter based microscopy and in vitro calbration as well as in vivo measurements were conducted -results sensors.

pHLourin2 protein supernatant with different pH values between pH 5.8 and 7.8. Analysis were performed using the Infinite M2000 pro Tecan plate reader with dual excitation at 405/5 nm and 485/5 nm and emission at 535/25 nm.