Difference between revisions of "Part:BBa K3866032"

Revision as of 09:18, 8 October 2021

N20bglΧ GB compatible with B3-B5

This is the E. coli β-glucosidase gene bglX with N20 signal peptide instead of its native signal peptide. It is based upon BBa_K523002.

Figure 1. The level 0 module : pupd2- N20bglx (illustration from SnapGene)

Usage and Biology

Bglx belongs to the family of β-D-Glucosidases. These enzymes catalyze the hydrolysis of cellobiose (1,4-β-D-glucopyranosyl-D-glucopyranose) into two molecules of glucose. The bgIX gene is located adjacent to the dld gene at 47.8 min or 2225 kb on the E. coli chromosome. The presence of a 20 amino acids signal peptide (at the N-terminal) is consistent with the periplasmic location of the mature protein. The replacement of native signal peptide with N20- signal peptide is expected to make the mature protein an extracellular product.

Design Notes

In order to construct this part, we replaced the native signal peptide with the N20 signal peptide sequence which is: 5’-atggaaggcaacacccgcgaagataactttaaacatctgctgggcaacgataacgtgaaacgcccgagcgaagcg-3’. The coding sequence was domesticated. We removed BsmBI, BsaI, BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is present in pUPD2 BBa_K3505007 and has overhangs compatible for GoldenBraid cloning. The CDS has position B3-B5.

Figure 2.The overhangs of this part in the GoldenBraid Grammar

Verification of cloning

Figure 3. (C= Cut, U=Uncut) Restriction digestion of pUPD2-N20-bglX (C1/U1) with: HindIII, Expected bands:3301bp, 1121bp, Positive result: C1

Experimental Use and Experience

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 without a carbon source (-)

Figure 5.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 6. 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 7. 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

According to first diagram, engineered bacteria with N20-signal peptide are able to grow, so they can use cellobiose as a carbon source. According to second diagram, engineered bacteria with N20-signal peptide have a higher growth rate than the bacteria with native signal peptide- bglX. This means that the first one can utilize both carbon sources, cellobiose and glucose, in a better way.

Sequence and Features

Source

Synthesized by IDT.

References

• Gao, D., Wang, S., Li, H., Yu, H., & Qi, Q. (2015). Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli. Microbial cell factories, 14, 49.
• Yang, M., Luoh, S., Goddard, A., Reilly, D., Henzel, W., & Bass, S. (1996). The bgIX gene located at 47.8 min on the Escherichia coll chromosome encodes a periplasmic -glucosidase. Microbiology, 142(7), 1659-1665.