Composite

Part:BBa_K523014

Designed by: Sylvia Ispasanie, Mun Ching Lee, Eugene Fletcher   Group: iGEM11_Edinburgh   (2011-09-08)
Revision as of 13:25, 13 September 2011 by Allancrossman (Talk | contribs) (→‎Experiment)

Plac + LacZ + bglX

The E. coli periplasmic β-glucosidase gene bglX under the control of the lac promoter. The native ribosome binding site is present.

Usage and Biology

The BglX β-glucosidase is expected to be localised to the periplasm. It ought to be capable of breaking down cellobiose.

Experiment

We conducted two assays, comparing the activity of this part (Plac-bglX) with that of the exoglucanase cex (BBa_K118022) which we also placed under the control of the lac promoter (yielding BBa_K523016) on two different substrates:

  • 4-methylumbelliferyl-beta-D-glucuronide (MUG, left photo). This substrate mimics cellobiose.
  • Something else (MUC, right photo). This substrate mimics cellulose.

Both substrates produce a fluorescent product when cleaved. Our plates below shows the results of placing cell lysate and cell debris on an MUG plate. Present on both plates are:

  • Left side of plate: JM109 expressing this part, K523014
  • Right side of plate: JM109 expressing exoglucanase cex, BBa_K523016
  • Bottom of plate: JM109 cells
BglX-MuG.jpg     Cex-MuC.jpg
MUG assay. bglX on left, cex on right.     MUC assay. bglX on left, cex on right.

As can be seen, bglX is capable of degrading MUG (the cellobiose mimic) while exoglucanase displays much weaker activity. By contrast, exoglucanase is much better at degrading MUC.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 607
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2266
    Illegal AgeI site found at 2488
    Illegal AgeI site found at 2677
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None