Difference between revisions of "Part:BBa K4621153"

Revision as of 18:06, 5 October 2023

It consists of the LPMO hypothetical promoter and EctOperon.

Usage, Biology and Characterization

EctOperon contains ectA, ectB, ectC and ectD and can produce 5-Hydroxyectoine from lysine.

LPMO hypothetical promoter is a whole functional sequence in front of the Lytic polysaccharide monooxygenase LPMO gene, which was cloned from SCUT-3 gene. In our project, through transcriptome analysis, qPCR and other means, we found that the gene expression of monooxygenase LPMO was up-regulated when SCUT-3 was in chitin medium, and we speculated that its promoter Pro lpmo may have properties that can be induced by chitin.

Testing and validation

We ligated the LPMO hypothetical promoter with EctOperon and overexpressed it in Streptomyces SCUT-3 using an integrated plasmid. The modified strain was inoculated in 40 mL LB medium and fermented at 40℃ and 220 rpm for 120h. The production of Ectoine and 5-Hydroxyectoin in the presence or without chitin (the first day of fermentation) was detected to evaluate the chitin inducability of the promoter. The results are shown in FIG. 1.

Fig.1 Fermentation of products with or without the addition of an inducer（chitin) (a).Ectoine (b).5-Hydroxyectoin(72h)

By adding chitin at different days after fermentation, we also determined that the optimal time for chitin induction of LPMO hypothetical promoter was the second day of fermentation, and the specific experimental data are shown in FIG. 2:

Fig.2 Fermentation of the product of SCUT-3-EctP at different induction times

Sequence and Features