Part:BBa_K1970000:Experience

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team:NTNU 2016

We tested this part together with an mCherry reporter ([1]) controlled by a self-inhibiting RBS targeted by this retron. We used IPTG and tetracyline to control the transcription of the two retrons and we measured the fluorescence of mCherry to record the output of this logic gate.

E. coli BL1-blue cells were transformed with the constructs above. Transformants and non-transformed bacteria (controls) were incubated for 2 hours in LB medium with amipicillin and chloramphenicol, at 37 °C and 255rpm. They were then transferred into 96 well plates and induced in groups of 4 replicates by all combinations of 3 IPTG and 3 tetractyline concentrations (one of which in both cases was zero). This gave 4 (replicates) x 9 (combinations) x 2 (transformant and control) wells. Time courses of the fluorescence (abs: 580nm, em: 615nm) of all wells were recorded over 5 hours in total (at 30 °C). After this 100 ul LB medium was added to each well and the measurements were repeated for 5 more hours. The resulting changes in fluorescence can be seen below (the upper two pictures are the transformants (left) and controls (right) during the first 5 hours of measurements, the lower to are similar but during the last 5 hours).

The colors above represent the averages over the 4 replicates for the 9 inducer combinations. The relative difference between these averages and the average for the uninduced wells (lower left corner) is found, and the differences between these values for the first and the last recording (5h and 0h) are plotted. We thus plot the growth of fluorescence in the induced wells relative to growth in uninduced ones.

The readings depend on the growth stage of the bacteria, with non-exponentially dividing bacteria (lower pictures) showing a pattern exhibiting an XOR-gate like response. Bacteria in the exponential phase show a non-XOR response (upper prictures). In either case the fluorescence response is relatively weak (<0.3 relative to uninduced cells) but significant.

User Reviews

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