RBS + bglX (E. coli perisplasmic β-glucosidase)

This is the E. coli β-glucosidase gene bglX. The part contains the native Ribosome Binding Site.

The part was made using the strategy outlined in BBa_K523000, and therefore contains 4 extra bases at the 5' end which generate a BglII restriction site.

Usage and Biology

The product protein is believed to be periplasmic. β-glucosidase cleaves β(1→4) bonds, i.e. those found in the disaccharide cellobiose.

The SignalP program predicts that a 20 amino acid localisation signal (at the N terminal) is cleaved off before the protein reaches its mature form.

Edinburgh 2011 carried out some experiments on this part under the control of the lac promoter - see details at part BBa_K523014.

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 AgeI site found at 1658
Illegal AgeI site found at 1880
Illegal AgeI site found at 2069
1000
COMPATIBLE WITH RFC[1000]

Functional Parameters: Austin_UTexas

BBa_K523002 parameters

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 BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, 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.

Thessaly 2021 Improvement

Experimental Use and Experience

This year we decided to improve BBa_K523002, by altering the native signal peptide sequence of BglX, a periplasmic beta glucosidase, with a N20- signal peptide, which translocates the protein extracellularly. With this improved part BBa_K3866032 , we engineered bacteria that are able to valorize cellobiose and use it as a carbon source.

Figure 1. Growth (OD600) of E. coli MC1061 cells transformed with N20-BglX (N20), native-peptide BglX (SP) or an empty vector (EMPTY), in a minimal M9 medium without a carbon source (-)

Figure 2.Growth (OD600) of E. coli MC1061 cells transformed with N20-BglX (N20), native-peptide BglX (SP) or an empty vector (EMPTY), in a minimal M9 medium with Cellobiose as a carbon source (CB).

Figure 3. Growth (OD600) of E. coli MC1061 cells transformed with N20-BglX (N20), native-peptide BglX (SP) or an empty vector (EMPTY), in a minimal M9 medium with Glucose and Cellobiose as a carbon source (2).

Figure 4. Growth (OD600) of E. coli MC1061 cells transformed with N20-BglX (N20), native-peptide BglX (SP) or an empty vector (EMPTY), in a minimal M9 medium with Glucose as a carbon source (GL).

Conclusion

N20-bglX bacteria grow more and faster than the SP-bglX and EMPTY bacteria in a 24-hour time-frame and with cellobiose as the only carbon resource. The engineered enzyme provides an advantage in situations where cellobiose is the only abundant carbon source. Also, N20-bglX bacteria reach an exponential-like phase much quicker than the native peptide or the empty vector control bacteria, which means quicker valorization cellobiose and update of glucose by the cell.

UMA_MALAGA 2022 Improvement

This year we decided to improve BBa_K523002 by deleting the native RBS and optimizing the whole sequence. We used our new part(BBa_K4368002) to have an enzymatic complex named CAZymes with cenA and cex to degrade cellulose into glucose.

Characterization

The expression cassette sequence was digested with EcoRI and PstI enzymes and subsequently ligated with a chloramphenicol resistant plasmid backbone (Cm). Transforming bacteria were created with this plasmid and seeded on LB-Agar+Cm plates. After growth, colonies were selected based on their color (white) and DNA extraction was performed using the Promega PureYield Plasmid Miniprep System kit. The resulting DNA is used for further digestion with EcoRI and PstI. The digests are then run on a 0.75% agarose gel at 90 mV voltage and constant amperage. BioRad brand RedSafe is used as an intercalating developing agent.

Enzyme digestion

Part:BBa_K523002