Difference between revisions of "Part:BBa K1689015"

Revision as of 00:32, 19 September 2015

F[1,2]-dCas9

F[1,2] segment of DHFR fused with dCas9

Dihydrofolate reductase(DHFR) exists in all kinds of organisms, which consists the adenine-binding domain (fragment [2]) and a discontinuous domain (fragment [I]/[3]). Fragments [1] and [2] contain folate-binding pocket and the NADPH-binding groove while fragment [3] has few crystal contacts with the substrates. DHFR catalyzes the NADPH-dependent reduction of dihydrofolate (H2folate) or folic acid to tetrahydrofolate (H4folate), tetrahydrofolate and its derivatives are essential for purin and thymidylate synthesis, necessary for cell division and growth. DHFR can be used for proetin fragment complementary assay(PCA), only reconstituted DHFR in the presence of inducer allows cell growth. The activity of DHFR can also be determined spectrophotometrically , with absorbance change at 340nm.

Here we choose to cleave EcDHFR at position 88 so as not to disrupt the active site and NADPH cofactor-binding sites, with the reduction of dihydrofolate (H2folate) or folic acid to tetrahydrofolate (H4folate), we could detect electrochemical signals immediately.

References:
1 Aiso, K., Nozaki, T., Shimoda, M., & Kokue, E. (1999). Assay of dihydrofolate reductase activity by monitoring tetrahydrofolate using high-performance liquid chromatography with electrochemical detection. Analytical biochemistry,272(2), 143-148.
2 Bystroff, C., & Kraut, J. (1991). Crystal structure of unliganded Escherichia coli dihydrofolate reductase. Ligand-induced conformational changes and cooperativity in binding.Biochemistry, 30(8), 2227-2239.

Sequence and Features