Difference between revisions of "Part:BBa K523014"

Latest revision as of 01:05, 4 September 2020

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

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.

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. 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

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

Functional Parameters

Functional Parameters: Austin_UTexas

Burden Imposed by this Part:

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This part exhibited a significant burden. Users should be aware that BioBricks with a burden of >20-30% may be susceptible to mutating to become less functional or nonfunctional as an evolutionary consequence of this fitness cost. This risk increases as they used for more bacterial cell divisions or in larger cultures. Users should be especially careful when combining multiple burdensome parts, as plasmids with a total burden of >40% are expected to mutate so quickly that they become unclonable. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.