Team:UANL Mty-Mexico/Wetlab

We divided our circuit in sub-circuits, or modules. Each module comprises a single function of our system. These modules are:

• The mCherry switch.- this switch comprises the 37°C thermometer and an mCherry reporter right downstream from it.
• The GFP switch.- this switch is similar to the mCherry one, but has a 32°C thermometer and a GFP reporter.
• The LacI-GFP switch.- in this switch, a 37°C thermometer is regulating the expression of a LacI gene, which in turn is regulating the expression of a GFP reporter. This GFP reporter is also under the regulation of a 32°C thermometer. In this way, we expect to see two different states: OFF at temperatures below 32°C; ON when temperature is between 32°C and 37°C; and OFF again when temperature is above 37°C.
• The TetR-mCherry switch.- here, an mCherry reporter is regulated by a 37°C thermometer and a pTet promoter; this switch also includes a TetR construction.
• The cI-TetR-mCherry switch.- this switch is similar to the previous one, but also includes a cassette that expresses a thermolabile version of cI. In this way, the expression of mCherry will be ON only at temperatures between 37°C and 42°C and OFF at other temperatures.

Fluorescence Assay

The part BBa_K110006 which has mCherry under the regulation of the TetR and the RNA Thermometer specific for the 37°C. To verify the operation, a series of experiments were developed in which the bacteria with this part were exposed to different temperatures under the following protocol.

1.- The synthetic constructions were transformed in DH5-alpha and planted in Petri dishes with LB Agar and the corresponding antibiotic.

2.- Twenty different clones were chosen and planted in test tubes with 3 mL and LB medium. They were incubated at 37°C to overnight until saturation.

3.- A visual section of the clones with more and less expression of the Red Fluorescent Protein (mCherry ) was made.

4. 20 μL of the cultivation of all the night were transferred to eppendorf tubes in the microcentrifuge with 500 μL of LB medium and antibiotic. A tiny hole was made to the cap of these tubes with a needle to allow the oxygenation of the cultivation over the experiment.

5.- They were placed at the thermomixer, in eppendorf adjusting the temperature to 25, 37 and 42°C and shaken at seventeen hours at 900 rpm.

6. 200 μl were took from each cultivation and placed in a black 96 well plaque Costar to make measurements in a fluorometer Biotech Synergy HT with the following conditions for mCherry. Excitation filter of 530 +/- 25nm, emission filter of 590 +/- 35 nm, sensibility of 85.

7. The optical density was determined for each cultivation to normalize the measurements reading in a Petri dish with transparent 96 well plaques Costar, with a wave length of 630 nm.

8. The data was processed with Excel.

Fluorescence assays results

Of 20 different clones derived from the transformation of E. coli DH5a with the synthetic part BBa_K1140006, four clones were selected based on their differences in colony color intensity.

We called the clones M1, M2, M11 and M12. M1 and M2 showed higher color intensity, while M11 and M12 showed less or no color. At least three independent experiments were performed incubating the samples in microcentrifuge tubes at 25, 30, 37 and 42°C for 17 hours. Fluorescence was measured for each sample and normalized with the OD of each bacterial culture. At 25ºC, most clones showed some leakiness.

Test at 30°C.

Similarly, at 30ºC the thermometer's structure should remain stable. However, the three clones showed a slight increase in red fluorescence (Fig. 3).

Test at 37°C.

Rising the temperature to 37ºC increases mCherry's expression in all three clones with different intensity. Theoretically, at this temperature the structure of the thermometer is completely open. Nonetheless, expression varies significantly among clones. M1 shows nearly a 10-fold increase in expression relative to that of 25°C. On the other hand, M2 is increased 7X while M11 is increased only 2X (Figure 4).

Test at 42°C.

Increasing the culture temperature to 42ºC does not have any significant effect, which agrees with the assumption that the thermometer retains an open structure (Figure 5) .

The M12 clone. One of the clones was selected for apparently having very low expression of mCherry, although it still proved to posses the construction. This was tested at the same time with the clones M1, M2 and M11. Figure 6 shows a comparison between M1 and M12 cloning at all three temperatures. Clearly the clone 12 does not have the behavior of the other three clones.

When the three clones are compared at all three temperatures, M1 and M2 show a similar behavior, while M11 has a slightly decreased expression. M12 clone did not show any difference in response to temperature changes. This clone (M12) will be sequenced to find the cause of this behavior. A likely explanation is a mutation preventing the expression of mCherry (Figure 7).

Plasmid copy number

We suspected variations in plasmid copy number (PCN) to be responsible for phenotypic differences among clones, which is usually estimated by southern blot or qPCR. However, as we did not have the reagents or equipment available to carry out such protocols, we developed a very simple and rapid methodology to look into this possibility as illustrated in the diagram below (figure 8).

This experiment was carried out as a superficial inquiry only. Because there is large variation in the DNA yield obtained through plasmid minipreparation, this protocol is in no way conclusive; however it gives us an idea of what is happening inside cells.

The chart on the left shows that, even if there exists a large variation among repetitions, M12 clone appears to have consistently smaller plasmid concentrations than M1 and M2 (figure 9). To support the existence of at least two statistically significant different groups (M12 vs M1 and M2), we conducted a one-way ANOVA test. The results are shown below.

Plasmid copy number Statistical analysis

Data Entry

*Plasmid DNA concentration by clones (ng/uL)

Data Summary

ANOVA Summary

*Independent Samples k=3

Tukey HSD Test

HSD[.05] = 724.74; HSD[.01] = 942.77

*M1 = mean of Sample 1, M2 = mean of Sample 2, and so forth.

HSD = the absolute [unsigned] difference between any two sample means required for significance at the designated level. HSD[.05] for the .05 level; HSD[.01] for the .01 level.

We found that values from M1 and M12, and M2 and M12 to be significantly different (p<0.05). On the other hand, M1 and M2 are not. This conclusion agrees with the impression given by the chart in figure 9. This results encourage us to further investigate PCN as the potential cause of phenotypic variations among clones.

The 37ºC RNAT proved to regulate expression in response to temperature changes. However, different outcomes were observed among clones, suggesting factors other than RNAT sequence influence reporter's expression. An exploratory assay points at plasmid copy number as the potential cause for this behavior, and the existence of at least two distinct groups was statistically proved. Further tests will be carried out to determine with higher accuracy the influence of plasmid copy number on the phenotypic variation observed among clones.

Overall, we were able to select a functional RNAT that produced nearly a 10-fold increase in expression at 37ºC relative to 25ºC. These results are encouraging as they prove a functional behavior of a crucial module in our genetic circuit. Further tests should be carried out in order to reproduce the 32ºC RNAT behavior in our construction, and finally test if their integration into our circuit results in a functional combination of transcriptional and translational regulation.