Part:BBa_K1547005
PSAD Promoter
This is a plant promoter from the green algae Chlamydomonas reinhardtii. It is a high expression promoter that encodes for a ferrodoxin-binding protein of photosystem I [1].
This PSAD promoter was characterized when we transformed our PSAD-GFP-NLS cassette (Bba_K1547001) into chlamydomonas reinhardtii (UVM1, and subsequently was analyzed via fluorescent microscopy.
Our GFP-NLS cassette, containing a strong PSAD promoter [2] and its respective terminator, was introduced into chlamydomonas reinhardtii (UVM1) by electroporation. Characterization was carried out after transformants were then grown in antibiotic selection media in 12:12 light/dark cycles, on a shaker, for several days. Fluorescent microscopy was used to examine the localization, and intensity of the GFP. Our preliminary images produced clear images of bright green areas in the transformant cells, whereas the wildtype cells lacked any bright areas. Moreover, the GFP appeared to be localized to one specific region in the cells. We can therefore conclude that our GFP-NLS construct produces a GFP that localizes to a specific location in chlamydomonas reinhardtii cells. Further DNA staining techniques can be used to verify that this region is the location of the nucleus, and further characterize our part in that manner. Images can be seen on Concordia 2014's wiki: http://2014.igem.org/Team:Concordia/Notebook . Furthermore, we noted that the intensity of our PSAD-GFP-NLS sample appeared much higher than that of our GFP construct controlled by an HSP70/RBCS2 fusion promoter.
Future Research can involve quantitative characterization using FLOW cytometry, with a correction for the autofluorescence of c. reinhardtii cells.
Transformant c. reinhardtii cells containing GFP, localized to a specific region:
Wildtype c. reinhardtii cells
1. Fuhrmann et al. (1999) Plant J. 19, 353-361
2. Harris, Elizabeth H. (2009). The Chlamydomonas Sourcebook (2nd Edition) Volume 1: Introduction to Chlamydomonas and Laboratory Use. Durham, North Carolina. Elsevier.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References [1] Fischer N, and Rochaix JD. (2001) The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Genet Genomics. 2001 Jul;265(5):888-94.
| None |