FRET Response of a Modified Ribose Receptor Expressed in the Diatom Thalassiosira pseudonana

Miller, Hanna

Pacific Northwest National Laboratory (U.S.)

关键词: Fluorescence;    Ribose;    Functionals;    Enzymes;    59 Basic Biological Sciences;   

DOI  :  10.2172/1033462
RP-ID  :  PNNL-20664
RP-ID  :  AC05-76RL01830
RP-ID  :  1033462

美国|英语

来源: UNT Digital Library

【 摘 要 】

The ability to insert complex proteins into silica has many applications including biosensing. Previous research has demonstrated how to direct proteins to the biosilica of diatoms [1]. Here, we show that a complex fusion protein that includes an enzyme, a bacterial ribose periplasmic binding protein, flanked by fluorescent proteins constituting a FRET pair can remain functional in the frustules of living diatoms. A Sil3 tag is attached to the N-terminal end to localize the fusion protein to frustules of the diatom Thalassiosira pseudonana. When ribose was applied, a larger decrease in FRET response was seen in transformed cells than in untransformed cells. Multiple forms of the expression vector were tested to find the optimal system; specifically, a one-vector system was compared to a two-vector system and the gDNA version of the Sil3 localization tag was compared to the cDNA version. The optimal system was found to be a one-vector system with the genomic version of the Sil3 tag to direct the protein to the frustules. Localization of the enzyme to the frustules was further confirmed through cell fluorescence imaging.