4 Notes
- Fusions can be generated in two main ways. One method is to
clone fragments of DNA into a plasmid vector that contains a
promoterless reporter gene(s) downstream of the multiple
cloning site. This library of fusions is then transformed or
electroporated into your organism of choice [40]. Plasmid-
based fusions can behave oddly based on their high copy num-
ber but they are easy to recover and characterize. If the vector
has conditional replication it can be used to generate fusions in
the chromosome by Campbell recombination (single cross-
over) [41]. In this report, we utilize another method of making
chromosomal fusions: random transposon integration. - There are numerous choices of transposon [42]. The transpo-
son should have random integration characteristics and a
selectable marker that functions in your organism of choice.
It should also create transcriptional fusions to a suitable
reporter gene(s). In this report we are using an mTn5 encoding
kanamycin resistance that creates fusions to theluxCDABE
operon ofPhotorhabdus luminescens[43]. - Transposons can be delivered as a purified protein complex by
electroporation (a transposome) [44], or delivered as a genetic
construct on a plasmid with conditional replication (a suicide
vector) either by electroporation, transduction, or mobiliza-
tion. In this report, we are using mobilization in which the
donor strain encodes the conjugation machinery. The suicide
vector has an RP4 origin of transfer (oriT) allowing passage
through the RP4-derived conjugative apparatus. The vector
has an R6K origin of replication lacking thepirgene that is
required for replication [45]. Thus, it can only replicate in host
strains that provide thepirgene [46]. Upon arrival in the
recipient strain the plasmid will fail to replicate. - Your recipient strain needs to have a characteristic that is not
present in the donor strain, i.e., a metabolic capability or anti-
biotic resistance. For this reason, most donor strains have
auxotrophies so that a recipient strain can be distinguished by
simply being prototrophic. Alternatively, your recipient strain
can encode an antibiotic resistance that is not present in the
donor strain. In our example, we selected for a spontaneous
resistance to nalidixic acid in our recipient strain by plating on
agar containing nalidixic acid. - In this chapter LuxR solos or orphans are identified in the
context of AHL signals. Therefore genomes lacking a cognate
LuxI for a LuxR would be classified as carrying a LuxR solo.
However, these LuxR solos might bind to novel non-AHL
signals, and once these new signals are identified and verified
Solo/Orphan QS Receptors 155