Composite

Part:BBa_K3866037

Designed by: Efthymia Zisopoulou   Group: iGEM21_Thessaly   (2021-09-27)


pAndersonJ23115:RBS-N20bglx-terminator

N20-bglX BBa_K3866032 under control of a constitutive promoter BBa_K3505012.

Fig.1: N20-bglX under control of a constitutive promoter.


Usage and Biology

Τhis part consists of AndersonJ23115 promoter BBa_K3505012, N20-bglX BBa_K3866032 and Double Terminator BBa_K3505017. By this way, as AndersonJ23115 is a strong constitutive promoter, bglx with the N20- signal peptide is constitutively expressed. The product protein is expected to be extracellular.

Design Notes

Τhe coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in alpha1R vector BBa_K3505008and has overhangs compatible for GoldenBraid cloning.

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
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 11
    Illegal NheI site found at 34
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1700
    Illegal AgeI site found at 1922
    Illegal AgeI site found at 2111
  • 1000
    COMPATIBLE WITH RFC[1000]


Source

Synthesized by IDT.

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