Composite

Part:BBa_K782063

Designed by: Fedja Pavlovec   Group: iGEM12_Slovenia   (2012-09-20)
Revision as of 17:45, 23 September 2012 by Anja Golob (Talk | contribs)

CMV promoter_mouse guanylate kinase_thymidine kinase 30

Introduction

Suicide gene therapy or gene-directed enzyme prodrug therapy (GDEPT)is widely used in cancer treatment. One of the most studied GDEPT systems is the herpes simplex virus thymidine kinase (HSV-TK) with purine nucleoside analog ganciclovir (GCV) as a prodrug. Systemic administration of the prodrug ganciclovir induces apoptosis only in cells transfected with HSV-thymidine kinase while the untransfected cells survive. Unlike human thymidine kinase, HSV-thymidine kinase is able to phosphorylate ganciclovir to form ganciclovir-monophosphate, which is then phosphorylated to ganciclovir-diphosphate and then to ganciclovir-triphosphate. Ganciclovir-triphosphate is then incorporated into DNA, which causes inhibition of DNA synthesis and subsequently leads to apoptosis (Ardiani et al., 2010). Following the BioBrick Standard Assembly technique we inserted the mouse guanylate kinase – thymidine kinase fusion gene (mGMK_TK30, (BBa_K404113)) under the control of CMV promoter. HSV-TK mutant (TK30) contains six amino acid substitutions and shows enhanced sensitivity to ganciclovir (Kokoris et al., 1999), while mouse guanylate kinase improves phosphorylation of ganciclovir-monophosphate to ganciclovir-diphosphate, thus preventing accumulation of ineffective intermediate products (i.e. GCV-MP, GCV-DP) due to limited ability of endogenous guanylate kinase..


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1315
    Illegal NgoMIV site found at 2220
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 707


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