Here is our engineering cycle!!
Design: The literature provides sequence but it contains illegal restriction sites so the sequence is modified to remove the sites and then optimized for use in E. coli.
Build:The part was built by IDT as a plasmid and then transformed into E. coli.
Test: We ligated the coding sequence to a stop sequence and successfully transformed into E. coli. We then ligated that to a strong constitutive promoter but the transformations failed multiple times.
Learn: Additional research showed that the intermediates in this pathway can be used as antimicrobials in some systems. Constitutive expression may be killing the transformed cells.
Redesign: Pull low level constitutive promoters, arabinose-inducible promoters, and osmotic pressure-inducible promoters to retry. This is currently in progress.