Due to restrictions imposed by the COVID-19 situation we were unable to perform any experiments. Protocols herein will be an indication of what we would aim to do if we had access to laboratories.
The goal of our synthetic biology plan was to engineer E. coli to produce salinomycin and then process salinomycin to produce narasin, an antiparasitic compound. This would complement our early detection system for amoebic gill disease (AGD) with a treatment option that is less harmful for fish in early steps of AGD. Our first step would be to clone the LuxR- family transcriptional regulator, SlnR into E. coli cells.
How would we reach that goal?
Our plan would be to clone the salinomycin producing gene cluster from Streptomyces Albus into E. coli . Since salinomycin targets gram-positive bacteria, the gram-negative E. coli is a good candidate for salinomycin production. Our first steps would be to isolate SlnR from Streptomyces Albus gDNA ( protocols 1.1 -1.6). We would then clone it into a pBAD/His A expression vector and transform it with chemically induced E. coli cells (protocols 2.1 - 3.1).
We would then aim to verify successful transformation via colony PCR, sequencing of transformants (protocols 3.2 - 3.4). To verify functional insert we would perform expression and purification of protein of interest and electrophoretic mobility shift assay[1,2] (protocols 4.1 - 4.2)
Articles that we drew inspiration from
 Zhu, Z., Li, H., Yu, P. et al. SlnR is a positive pathway-specific regulator for salinomycin biosynthesis in Streptomyces albus . Appl Microbiol Biotechnol 101, 1547–1557 (2017). https://link.springer.com/article/10.1007/s00253-016-7918-5
 Liu, S., Yu, P., Yuan, P. et al. Sigma factor WhiGch positively regulates natamycin production in Streptomyces chattanoogensis L10. Appl Microbiol Biotechnol 99, 2715–2726 (2015). https://link.springer.com/article/10.1007%2Fs00253-014-6307-1
Click to view our protocols in PDF-format.
- 1.1 Isolation of gDNA from Streptomyces albus
- 1.2 Amplification of SlnR from Streptomyces albus gDNA
- 1.3 PCR purification via QIAGEN PCR cleanup
- 1.4 Miniprep of pBAD_His from bacterial strain
- 1.5 Amplification of pBAD_His vector via Phusion PCR
- 1.6 DpnI Digestion
- 2.1 Gibson assembly of vector and amplified insert
- 2.2 Preparing chemically competent DH5α cells
- 3.1 Transformation of chemically competent cells
- 3.2 Verifying cloning with colony PCR
- 3.3 Extraction of plasmid DNA from DH5α cells using QIAprep Spin Miniprep Kit
- 3.4 Verification of insert via sequencing of transformants
- 4.1 Expression and purification of protein of interest
- 4.2 Electrophoretic mobility shift assay