Part:BBa_M50034:Experience

Stanford Location: Plasmid Name - U10 Thermosensor Construct; DNA2.0 Gene # - 273829; Organism - E. coli; Device Type - Sensor; Barcode # - 0133027185; Box Label - BIOE44 F16

Our unique parts are based off of E. coli thermosensors designed by Neupert, J., et al. We plan to utilize and quantify two of their thermosensors: U1 and U10, each of which has a different melting stability and hence unique temperature sensitivity. In our experiment, we used E. coli to test these sensors because of the bacterium’s viability in the temperature ranges of the RNA thermosensors, its ease of handling, and well-understood laboratory properties (see Figure 1).

Figure 1. RNA hairpin structure of U1 and U10 thermometers with calculated free energies2. U10 has a potentially higher melting stability than U1.

At elevated temperatures, the U1/U10 thermosensor releases its hairpin structure in the 5’-UTR and allows the RBS to be readily accessible to the ribosome, enabling increased expression of our desired gene (see figure 2).

Figure 2. Plasmid schematics outlining our group’s designed and synthesized plasmids: pU1_mTurquoise2 and pU10_mKO2. Includes 5'-UTR, FP, and selective antibiotic.

The U1 thermosensor detects temperatures ranging from 22˚C to 37˚C and exhibits the property of more gene expression at higher temperatures. In contrast, the U10 thermosensor mainly allows gene expression at 37˚C, while 30˚C and 22˚C show very low and similar gene expression levels. U10 has a higher specificity as a thermosensor for 37 ˚C than U1 in the temperature range of 22˚C to 37˚C. We did not make any modifications to the thermosensor genetic sequences given by Neupert, J., et al. We decided to use mKO2, mTurquoise2, and Clover FPs for our constructs so that all three FPs can be expressed in a single E. coli cell line without interfering with each other in a fluorescence assay, as these FPs have distinct excitation/emission wavelengths. These proteins are distinct from one another by their λex and λem, and were expected to provide accurate, distinguishable measurements of expression activity across the temperature gradient. However, because the plate reader limits to a minimum wavelength difference of 30 nm between λex and λem, we had to adjust our experimental protocol to accommodate this requirement (see Table 1).

Table 1. The optimal/experimental λex and λem for our three FPs: mTurquoise2, mKO2, and Clover. Also included is the gain we used for plate reading.

OD600/FP Endpoint Assay Blank measurements were done using 500µL of LB in separate wells. The 96-well plate, cultures, and dilutions of cell cultures were all kept on ice to prevent temperature specific expression at room temperature. The plate was then transferred to an incubator set at 25˚C for 48 hours or incubated at 30˚C or 37˚C for 24 hours and shaken at 225 rpm. The additional 24 hours intended to compensate for slower cell growth at low temperatures. We increased our lower bound from 22˚C to 25˚C because the incubators were more consistent in maintaining a temperature higher than the ambient room temperature. Therefore, all subsequent experiments including the kinetic assay were performed at 25˚C. After incubation at the experimental temperature, 200µL of the cell cultures in each well of the incubation plate was transferred to a sterile transparent 96 well plate, sitting on ice to prevent muddling the experimental data with unwanted protein synthesis. In a plate reader, the transparent plate was fluorescently analyzed at different wavelengths for each FP. Also, an OD600 was done on every well to get cell density measurements, which provided useful for normalizing fluorescence readings in our analysis. All OD600 and fluorescence measurement will be done on each plasmid row as well as the blank LB (control) row with no FP expression.

OD600/FP Kinetic Assay After the initial culture step, an OD600 absorbance reading was done and the cell culture was diluted to reach an absorbance of approximately 0.1. Then 20µL of diluted cell culture were pipetted into the translucent 96 well plate, followed by 180µL of LB nutrient broth and 0.2µL of 1M IPTG. OD600 absorbance, mTurquoise2, mKO2, and Clover readings were done every 10 minutes for 10 hours, with the plate reader's internal temperature set to the temperature for that experiment and set to shake at 225 cpm.

Data Analysis For our OD600/FP endpoint and kinetic assay, we normalized our fluorescence to cell density with OD600 and LB blank with the equation Normalized FP= (FP sample−FP of blank)(OD600 sample−OD600 of blank)

For our OD600/FP endpoint assay, we performed an additional normalization so that every value is a fraction of the respective 37˚C mean to enable comparison between different FPs.

Results

OD600/FP Endpoint Assay

Our U1 and U10 thermosensors both showed increased expression at 37˚C. The U1-transformed cells showed a ~3 fold increase of mTurqoise2 expression at 37˚C compared to expression at 25˚C and 30˚C. The U10-transformed cells showed a ~5 fold increase of mKO2 expression at 37˚C compared to expression at 25˚C and 30˚C. Furthermore, the mean variance at 37˚C is outside the mean variance ranges at 25˚C and 30˚C, indicating a definite and substantial increase in FP expression. The fluorescence level at 37˚C had much higher variance compared to the variance of 25˚C and 30˚C (see figure 3).

Figure 3. Normalized fluorescence intensities for U1, U10, and Clover transformed cells at three different temperatures.

This may be due to the slight innate thermosensitivity of the RBS15 or small changes in FP activity at different temperatures.

Clover, which was intended to be a positive control, is a reliable source of comparison only at 30˚C and 37˚C. The Clover-transformed cells presented somewhat as expected at 30˚C and 37˚C. Clover expression decreased by approximately 25% at a lower temperature, but generally, expressed as we predicted (i.e. Clover expression stays relatively consistent). In addition, the mean variance at both 30˚C and 37˚C were relatively low indicating a reliable measure of Clover fluorescence.

Overall, U1 and U10-transformed cells showed a substantial increase of fluorescence at a temperature of 37˚C while leaving lower expression at a temperature of 25˚C and 30˚C. Using a LB blank as a negative control in the plate reader allowed for calculation of the normalized values which is what made any coherent analysis possible and for detecting possible bacterial contamination. Indeed, our positive control of Clover-transformed cells confirmed our findings since Clover fluorescence stays relatively stable across the temperature even with a slight decrease at lower temperatures.

OD600/FP Kinetic Assay

All assays running at 37˚C (U1_mTurquoise, U10_mKO2, and Clover) exhibited a decline in normalized fluorescence levels over the course of ten hours according to our data of OD600 and fluorescence readings. U1 peaked in fluorescence at about 3.5 hours at ~50,000 AFU/OD600 but declined steadily as the readings proceeded. U10 and Clover displayed almost identical fluorescence patterns at 37˚C as they show mostly a downward trend in normalized fluorescence levels (see figure 4).

Figure 4. 37 ̊C kinetic assay with normalized fluorescence for U1, U10, and Clover cells over a 10-hour period.

These results possibly indicate the dilution of fluorescent proteins over time as the cells divide or the bleaching of all fluorescent proteins after repeated exposure of a high-energy laser. There is not any data for the 30˚C kinetic assay. The 25˚C kinetic data did not truly represent the behavior at 25˚C, but rather at 27-28˚C. The plate reader did not reach all the way down to 25˚C. The trend for U1_mTurquoise, U10_mKO2, and Clover generally indicated a very slight, steady increase. In the best case, U1 seemed to increase by the largest margin by approximately 12%. U10 likewise seemed to increase in fluorescence by ~2 fold, hitting an observable peak at about 7.5 hours. Clover, our positive control, maintained a steady level of fluorescence and slight increased at the 7 hour mark (see figure 5).

Figure 5. 27 ̊C kinetic assay with normalized fluorescence for U1, U10, and Clover cells over a 10-hour period.

U1 and Clover did not seem to grow much at all, hovering around a OD600 of 0.15-0.18 over the 10 hour period.

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