Triple knockout strain

In order to use the orthogonal hydrolases as our reporter system in the sender-receiver set-up, we needed to use a strain of E.coli where the expression of native hydrolase genes were knocked out. This is to prevent background hydrolysis of hydrolases.
In our sender-receiver circuit, we use three hydrolases gusA , aes and nagZ. Hence, three hydrolase genes were knocked out of E.coli strain MG1655. The gusA hydrolase was knocked out by a scarless deletion scheme that left no scar sequences after the deletion method [1]. The subsequent deletions of aes and nagZ was carried out by the P1 phage transduction by using deletion strain from the Keio collection [2]. Thus we were able to use this strain MG1655△gus△aes△nagZ knocked out of expression of the three hydrolase genes in our project.

The triple mutant is used for the sender cells which are made to constitutively express nagZ. In the receiver cells, the hydrolases aes and gusA are expressed under the control of OHHL induced pluxR promoters.

References

1. Martinez-Garcia .E, de Lorenzo .V. 2012. Transposon based and plasmid based genetic tools for editing genomes of gram-negative bacteria. Methods in Molecular Biology. 813:267-283.doi: 10.1007/978-1-61779-412-4_16.
2. Thomason L.C., Costantino .N, Court .D .L, 2007. E.coli Genome Manipulation by P1 Transduction. Current Protocols in Molecular Biology.1.17.1-1.17.8.

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