Composite

Part:BBa_K523014

Designed by: Sylvia Ispasanie, Mun Ching Lee, Eugene Fletcher   Group: iGEM11_Edinburgh   (2011-09-08)
Revision as of 14:55, 1 November 2017 by Malta14 (Talk | contribs) (→‎E.coli in fluid media)

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.

MUG/MUC experiment

We (Edinburgh 2011) conducted two assays, comparing the activity of this part (Plac-bglX) with that of the exoglucanase cex under the control of the lac promoter (BBa_K523016) on two different substrates:

  • 4-methylumbelliferyl β- D- glucuronide (MUG, left photo). This substrate is a cellobiose analog.
  • 4-methylumbelliferyl β- D- cellobioside (MUC, right photo). This substrate is larger and is more like a cellulose analog.

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

  • Left side of plate: lysate/debris from JM109 expressing this part, K523014
  • Right side of plate: lysate/debris from JM109 expressing exoglucanase cex, BBa_K523016
  • Bottom of plate: lysate/debris from 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 analog) while exoglucanase displays much weaker activity. By contrast, exoglucanase is much better at degrading MUC.

Inability to grow on cellobiose

We tested the ability of E. coli with this part to grow on minimal media with cellobiose as the only carbon source. It could not. By contrast, it did grow if glucose was the carbon source, showing that it is fundamentally capable of growing on minimal media.


K523014 on cellobiose.jpg     K523014 glucose control.jpg
Cellobiose as the only carbon source. K523014 (bottom) fails to grow.     Glucose as the only carbon source. K523014 can grow.

E.coli in fluid media

(characterize by SDU-Denmark)

It was not possible to see any indications of E.coli being capable of living on cellobiose in fluid media either. On the figure below, it is shown that this biobrick can not make E.coli grow with cellobiose as the only carbon source. The bglX is compared to another biobrick which have the ability to degrade cellobiose to glucose cep94A/BBa_K2449004

T--SDU-Denmark--SD_Growth_experiment_bglx.jpg

Figure 3:Growth on cellubiose comparing BBa_523014/bglX to BBa_2449004/cep94A compared

Conclusion

This biobrick does not work as intended.

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]


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Categories
Parameters
None