Difference between revisions of "Part:BBa K1970000:Experience"

Revision as of 13:12, 26 October 2016

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Applications of BBa_K1970004

User Reviews

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) well. 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 the same 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 that 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

UNIQ9d244edc75926587-partinfo-00000000-QINU UNIQ9d244edc75926587-partinfo-00000001-QINU

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 team:<b>NTNU 2016</b>
-===Applications of BBa_K608351===
+We tested this part together with an mCherry reporter ([https://parts.igem.org/Part:BBa_E1010]) 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.
-We tested this part together with an mCherry reporter ([https://parts.igem.org/Part:BBa_E1010]) controlled by a self-inhibiting RBS. The retrons in this part are set to produce msDNA that targets the inhibitory stem loop of this RBS thereby inducing translational activity from it (an thus production of mCherry). Additionally the
 <br>
-E. Coli DH5α cells were transformed with the constructs above. Transformants were inoculated overnight in LB medium with kanamycin, at 37 °C and 255rpm. The 5 cultures were placed in a water bath at 37 °C. The water bath was gradually heated to 42 °C to avoid heat shocking the cells. Time courses of the fluorescence (abs: 580nm, em: 615nm) and OD of the cells were recorded (see the figure below).
+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) well. 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 the same but during the last 5 hours).
 <br>
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 [[Image:24_10_2_fused.png|900px]]
-The four combinations of RBS and terminator around an mCherry reporter coupled to a CI repressor (this part). In a) and b) the RBS was clearly not effective, with the fluorescence being close to that of cells lacking an mCherry. The heat treatment did not have any obvious influence on the fluorescence of the cells. The RBS in c) and d) had relatively high fluorescence values, which changed by an order of magnitude with the terminator chosen. However, the heat treatment did not have any obvious positive influence in these cases either. In d) the influence was negative in fact.
+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 that growth in uninduced ones.
-<br>
-Therefore we concluded that this part is not a good choice for temperature transitions between 37 and 42 °C. This agrees with the experience gained on this part by other groups.
+<br>
+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===