Part:BBa_K1140006:Experience

Applications of BBa_K1140006

The team observed that this part works correctly in E. coli K12. The synthetic RNAT proved to regulate expression in response to temperature changes. Figures 1 and 2 show the visual appearance of cultures grown at 37ºC containing

Figure 1. Temperature dependence of mCherry translation by u6 RNA thermometer in E. coli. Tet repressor is NOT present in this test. Two control cultures without mCherry sequence are included for growth and color comparison. a) Control 30ºC b)Control 37ºC c)30ºC d)37ºC.

Figure 2. Temperature dependence of mCherry translation by 37ºC RNA thermometer in E. coli at 37ºC. a)37ºC RNAT mCherry b)Non-fluorescent control c)Standard constitutively expressing RFP.

Surprisingly, we obtained different behaviors in clones derived from the same DNA transformation (figure 3). All measurements were performed at least in triplicate, the aritmethic mean is shown.

Figure 3. Behavior of different clones transformed with this construction (M1, 2, 11 and 12). Relative fluorescence under 25, 30, 37 and 42 celcius grades in E. coli.

For each measure in a given temperature, the system was left until a point in which we were sure the O.D of the cell culture and the production of the protein were in equilibrium, steady, and uniform, before the cells population started to decrease (which we found was 17h). We took as a standard for the RFUs the amount of fluorescence emitted by an E. coli K12 culture transformed with a constitutively expressed part BBa_E1010 (the amount of fluorescence emitted by our culture was calculated by dividing the fluorescence of the sample by the fluorescence of the standard). Figure 4 shows the behavior of our best clone, dubbed M1. M12 clone, showing a weird behavior, is to be sequenced to verify if this outcome is due to mutation or intrinsic cellular noise.

Figure 4. Relative fluorescence under 25, 30, 37 and 42 celcius grades in E. coli. M1 is a group of cultures used by UANL_Mty-Mexico team. Tet repressor is NOT present in this test.

We suspected variations in plasmid copy number (PCN) to be the potential cause of phenotypic discrepancies among clones. To look into this possibility, we examined plasmid DNA concentration (yielded from plasmid miniprep) as an indirect measurement of PCN. Even if there exists a large variation among repetitions, M12 clone appears to have consistently smaller plasmid concentrations (figure 5), suggesting PCN could indeed be the cause of this behavior.

Figure 5. Plasmid DNA concentration by clones (ng/uL).

Mathematically, we found that a simple gaussian function fits our data well, and it provides us a way to quantify the strength (amplitude), optimal value (horizontal shift), and definition or clearness (width) of our RNAT activity (figure 6). We believe positive slope is due to RNAT melting, while negative slope is due to increase in the overall protein degradation rate due to higher temperatures. This function also allows for comparisons between different RNAT, as well as being potentially predictive for non verified temperatures.

Figure 6. Gaussian Function fitting of the experimental data shown in figure 3.

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