Part:BBa_K784010:Experience

Experience of BBa_K784010

Experimental setup

In order to provide quality information about the riboswitch function, we fused the MCS+tac promoter+RS construct from the pUC19 to the mCherry protein. The resulting construct was used in a plate reader assay to measure the level of fluorescence as a function of theophylline concentration. After consulting with Dennis Mishler from the Gallivan lab, we learned that full induction of translation should occur at 2mM of theophylline. Thus, we planned a dose response experiment with concentration ranging from 0-8mM theophylline. Several experiments were done, as described in the results section of this page.
During the second set of experiments, a negative and a positive control were introduced. The negative control is pSB1C3 with an MCS insert (BBa_K784023). The positive control is a plasmid in which the RS was deleted and replaced with a spacer region with the RBS from the riboswitch (BBa_K784011).
All plate reader experiments were done in E. coli TOP10 strain. Briefly, starters were grown over night, diluted 1:100, grown to ~O.D 0.6 and divided into 48 well plates. IPTG was added (to 1mM concentration) to induce the tac promoter and different concentrations of theophylline were added. Controls without IPTG and without theophylline were prepared as well. The samples were incubated at 37 degrees for ~3-4.5 hours before measuring fluorescence in the plate reader.

First experiment

Figure 1: mCherry fluorescence divided by culture O.D as a function of theophylline concentration after induction by IPTG. Each point is an average of a duplicate. The experiment was done in the pSB1C3 backbone. Measurement was done after 4.5 hours of induction.

The first set of experiments was done in pSB1C3, which is a high copy plasmid backbone. This set of experiments lacked a negative and a positive control. The fluorescence divided by the O.D. as a function of the theophylline concentration is presented in Figure 1. Each result is an average of a duplicate.

It can be seen that there was no increase in the fluorescence when the theophylline concentration was increased, in contrast to our expectations. Moreover, the fluorescence/O.D value of the no IPTG control was ~32000.
The intermediate conclusion at this stage was that the mCherry might be accumulating in the cell due to the high copy number of the plasmid. Since the TOP10 strain has only low level of lacI, the transcription from the tac promoter occurs continuously. Therefore, the basal translation level is enough for the mCherry to accumulate.

Second set of experiments

Figure 2: mCherry fluorescence divided by culture O.D as a function of theophylline concentration after induction by IPTG. Each point is an average of a duplicate. The experiment was done in the pSB1C3 and pSB3C5 backbones. The results of the positive controls are at 0mM theophylline and 1 mM IPTG. Measurement was done after 4 hours of induction.

The second set of experiments was done in pSB3C5, which was believed to be a low copy plasmid backbone, in addition to repeating the experiment in pSB1C3. This set of experiments did have a negative and a positive control, as described above. The fluorescence divided by the O.D. as a function of the theophylline concentration is presented in Figure 2. Each result is an average of a duplicate. The negative control has been deducted from all the results.

It can be seen that there was no increase in the fluorescence when the theophylline concentration was increased, in contrast to our expectations. Again, the no IPTG control gave similar results to 0mM theophylline. The positive control in pSB3C5 gives ~1.5 higher result than the RS results, as expected. However, the positive control in pSB1C3 gives ~1.5 lower result than the RS results. It should be noted that there is a slight difference in the sequence of the positive controls in the spacer region, due to mutations during the cloning process.
While working with the pSB3C5 backbone we noticed that it gave higher miniprep results than pSB1C3 which made us suspect that it is not low copy. Therefore, we decided to repeat the experiment in a verified low copy backbone from Roee, which was notated pCP.

Figure 3: mCherry fluorescence divided by culture O.D as a function of theophylline concentration after induction by IPTG. Each point is an average of a duplicate. The experiment was done in the pCP backbone. Measurement was done after 3.5 hours of induction.

Third set of experimetns

This final set of measurements was done in pCP. The inserts were taken from the pSB3C5 backbone from the previous experiment. This set of experiments did have a negative and a positive control, as described above. The fluorescence divided by the O.D. as a function of the theophylline concentration is presented in Figure 3. Each result is an average of a duplicate. The negative control has been deducted from all the results.

It can be seen that there was no increase in the fluorescence when the theophylline concentration was increased, in contrast to our expectations. Again, the no IPTG control gave similar results to 0mM theophylline. The positive control gave ~3 times lower results than the RS results.

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