Davidson College Department of Biology Honors Thesis Title ...

Fluorescence Measurements for the pLas Promoters
Because I had constructed a transgenic strain of E. coli that was capable of expressing LuxR
and LasR, it was now possible for me to test the functionality of the pLas’ and pLasLux
promoters that had been constructed over the summer. Constructs had been built previously
that contained GFP downstream of the pLas’ and pLasLux promoters (BBa S03981 and
BBa S03984). I put these constructs into transgenic cells and non-transgenic cells. I also
used wildtype MC4100 cells as a negative control and GFP producing construct (K091131)
in HB101 as a positive control. Cells were exposed to a range of the different autoinducers in
LB+antibiotic and fluorescence was measured after 20 hours of incubation at 37 ◦ C (Figure
23).
The two pLas promoters had very different responses to the various autoinducer inputs.
pLas’ demonstrated the expected expression pattern, as it was activated by 3OC12 and not
induced by 3OC6. Additionally, the promoter was only activated in the transgenic MC4100
strain, providing further evidence that the LuxR/LasR expression cassette is in fact in the
genome of the E. coli. The pLasLux promoter, however, appeared to be trasnscriptionally
silent for all autoinducer inputs. One possible interpretation of these results is that, contrary
to what we found in the literature (Waters and Bassler, 2005), 3OC12 was able to bind to
both of the modified las and lux boxes (with the help of either LuxR or LasR). This one-way
cross-reactivity could have repressed the pLasLux promoter at the lux box when only 3OC12
was supplied to the cells.
To test if cross reactivity existed between 3OC12 and the lux system, I utilized two existing
constructs, the Lux Receiver (BBa K09100) and Las Receiver (BBa K091134). These con-
structs express LuxR and LasR respectively (when activated with IPTG) and also contain
their respective wild-type promoter upstream of a GFP expression cassette (Figure 24). I
exposed each of these receiver constructs (and controls) to all autoinducer treatments and
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