Coding

Part:BBa_K3866032

Designed by: Efthymia Zisopoulou   Group: iGEM21_Thessaly   (2021-09-27)
Revision as of 08:12, 15 October 2021 by Igkoni (Talk | contribs) (→‎Experimental Use and Experience)


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

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.

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 1637
    Illegal AgeI site found at 1859
    Illegal AgeI site found at 2048
  • 1000
    COMPATIBLE WITH RFC[1000]

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.
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