Composite

Part:BBa_K137053

Designed by: Victoria Hsiao   Group: iGEM08_Caltech   (2008-07-28)

Constitutive folB expression construct

This is the folate synthesis gene folB, one of five genes involved in the folate synthesis operon. In this construct, folB has been inserted behind the strong promoter J23100, the strong RBS B0034, and in front of the double terminator B0015. The vector is the inducible copy plasmid pSB2K3, which can be induced to high copy via IPTG.

The purpose of this construct is to test the effects of FolB (dihydroneopterin aldolase) overexpression on total folate production in Escherichia coli, with the goal of increasing total folate production.


Usage and Biology

Using the Caltech iGEM microbiological folate assay, K137053 in pSB2K3 in DH10B produced the following results:

FolB.gif

Increasing the FolB expression level (by raising the plasmid copy number using IPTG) increased folate production (reflected as an increase in the absorbance). However, this effect disappeared on addition of PABA. Either the changes in folate levels were not reflected in changes in absorbance (perhaps the assay saturated) or the reaction catalyzed by FolB was no longer rate limiting under these conditions. In the latter case, we expect that coexpression of FolP (the enzyme which joins the pterin ring from FolBKE with the PABA moiety) would increase pathway flux.

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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