Part:BBa_K1970000
Deoxynucleotide ribonuclease expressing plasmid which cuts off self inhibitory RBS tails
This plasmid contains two genes that code for two separate deoxynucleotide ribonucleases. These are able to bind to the RBS end of a modified mCherry mRNA. The modified mCherry mRNA has a self inhibitory RBS tail, which prevents binding of the transcript to ribosomes. Cleaveage by one of the deoxynucleotide ribonucleases removes the self inhibitory tail from the RBS, freeing the transcript for translation by ribosomes. Additionally the ribonucleases bind and inhibit the activity of each other to enable a XOR-gate like behaviour.
The two ribonucleases are under the control of lacI and tetR promoter. Thus IPTG and tetracyline (or analogs thereof) can be used as logic gate inputs, while the mCherry fluorescence as output.
Usage and Biology
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.
An alternative representation of the lower left graph is shown below. The data for the corners of the chart are plotted with their corresponding errors.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 341
Illegal NheI site found at 1575 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 619
Illegal XhoI site found at 1853 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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