Part:BBa_K1898100

ch25h, cholesterol 25 hydroxylase

This DNA codes for ch25h, also known as cholesterol 25-hydroxylase, which catalyzes the formation of 25 hydroxycholesterol from cholesterol.

Usage and Biology

25 hydroxycholesterol (25 HC) has been reported to have a range of effects on the immune system, including the suppression of production of IgA by B cells and inducing the migration of human peripheral blood monocytes [1]. 25 HC is also found to be able to reverse protein aggregation and restore solubility to the proteins [2]. In our project, we aim to deliver CH25H to the lens, where 25 HC will be produced and reverse aggregated lens protein. Aged-induced cataracts occur when lens proteins get oxidized and clump together. The increase of 25HC in the lens will help to treat cataracts.

[1] Diczfalusy, U. (2013). On the formation and possible biological role of 25-hydroxycholesterol. Biochimie, 95(3), 455-460. doi:10.1016/j.biochi.2012.06.016

[2] Makley, L. N., McMenimen, K. A., DeVree, B. T., Goldman, J. W., McGlasson, B. N., Rajagopal, P., Dunyak, B.M., McQuade, T.J., Thompson, A.D., Sunahara, R., Klevit, R.E., Andley, U.P., and Gestwicki, J.E. (2015). Pharmacological chaperone for α-crystallin partially restores transparency in cataract models. Science, 350(6261), 674-677.

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
  COMPATIBLE WITH RFC[1000]

Gel Pictures

We designed primers to remove a stop codon and to move CH25H cDNA into iGEM BioBrick. PCR was set up and the gel picture is shown below. The expected size for CH25H PCR check is ~1.1kb. The bands boxed in red are the correct bands.

Sequencing

CH25H was sent to sequencing and the result shows that the CRYAA sequencing result is correct (with no stop codon):

The four cutting sites are highlighted in red and cryaa is highlighted in orange