Team:Rice/Results

RNA Thermometer Results

To test the existing kit thermometers and our computationally designed thermometers in vivo, we made an RNA thermometer testing circuit, transformed it into E. coli, and tested the fluorescent output at different temperatures. These graphs show the relative fluorescent intensity of each thermometer between either 25 °C and 30 °C or 25 °C and 37 °C. Only thermometers that performed better than the positive control were included. "K" stands for the kit thermometers, and "NC" stands for our computationally designed NoChill series of thermometers registered as a part. "V" thermometers are thermometers designed and tested but not registered. For additional thermometer testing data, please reference the contribution page.

As the first graph shows, most thermometers had about a two-fold change in expression between 25 °C and 30 °C. Additionally, most did not perform better than the existing kit thermometer. Since a 5 °C change in temperature is not that much, we also tested the difference between 25 °C and 37 °C, and the second graph shows that most of the thermometers in that experiment had around a four to six–fold expression change.

Figure 1a: The ratio of Fluorescence per OD between the temperatures of 30°C and 25°C. The horizontal dash line is used to compare the thermometers we made to that of the best performing kit thermometer.

Figure 1b: The ratio of Fluorescence per OD between the temperatures of 30°C and 25°C. The horizontal dash line is used to compare the thermometers we made to that of the best performing kit thermometer.

Figure 2: The ratio of Fluorescence per OD between the temperatures of 37°C and 30°C. The horizontal dash line is used to compare the thermometers we made to that of the best performing kit thermometer.

Figure 3: The ratio of Fluorescence per OD between the temperatures of 37°,30°C and 25°C.

Plant Growth Results

Determining Plant Media Conditions

The lab that helped us with our plant experiments grew Arabidopsis thaliana in PNS media. However, we wanted to make sure that our Pseudomonas putida would also be able to grow in the media, because it cannot use sucrose as the sole carbon source. We tested the effect of sodium, sodium citrate, glucose, and glucose+sodium on Pseudomonas growth. The bacteria grew in all conditions except for just sodium, so for ease, we decided to add glucose to our plant medium.

We needed to make sure that adding glucose would not adversely affect plant growth. To do this we tested the effect of two different glucose concentrations on plant and bacteria growth and found that the plants grew noticebly more at 0.01% glucose.

Plant Root Growth with only 1% PNS

General plant root growth protocols use 1% PNS, so we wanted to have a control experiment for plant root growth under general lab conditions, without any supplementation of any other sugars or hormones present in our enzyme circuits. Preliminary scans of the plates show sporadic root growth so we deemed the results unusable, and we are in the process of repeating these experiments.

Plant Root Growth with Arabinose

Plant root growth was tested with different concentrations of arabinose because arabinose is the inducer for our enzyme circuits. Since arabinose is a sugar that the plant could metabolize, it was necessary to see how different concentrations of arabinose would affect plant growth without our enzyme circuits. Preliminary scans of the plates show sporadic root growth so we deemed the results unusable, and we are in the process of repeating these experiments.

Plant Root Growth with Auxin (IAA)

IAA (auxin) is a plant hormone that stimulates plant growth and root elongation. Since one of our enzyme circuits will upregulate the production of IAA, it was essential for us to have a control experiment of plant root growth with IAA. This will later be used to compare root growth when we grow plants with our enzyme circuits. Preliminary scans of the plates show sporadic root growth so we deemed the results unusable, and we are in the process of repeating these experiments.

Plant Root Growth with Trehalose

Trehalose is a disaccharide produced in one of our enzyme circuits in order to facilitate plant growth and survival under hot and arid environments. Since trehalose on its own is a signaling metabolite for plant growth and protects plants against dessication stress, it was necessary to conduct a control experiment to measure how trehalose affects plant root growth before. Again, it control experiment will give us an idea of how trehalose affects plant root growth in comparison to when the plants are inoculated with our enzyme circuits. Preliminary scans of the plates show sporadic root growth so we deemed the results unusable, and we are in the process of repeating these experiments.

Plant Root Growth with Arabinose, Auxin, and Trehalose at Maximum Concentration

Our final circuit will have all three enzyme circuits working together to produce ACC deaminase, IAA, and trehalose. Since the inducer of these circuits (arabinose), IAA, and trehalose are likely to affect root growth, it is not only necessary to have control experiments for plant root growth with each separately, but also together to simulate the final circuit with all three working within the same system. Therefore, having a control experiment of arabinose, IAA, and trehalose at maximum concentration would give us the necessary comparison for when we test all three enzyme circuits together and their effects on plant root growth. Preliminary scans of the plates show sporadic root growth so we deemed the results unusable, and we are in the process of repeating these experiments.

Testing A. thaliana with induced and uninduced P. putida containing enzyme constructs

All plates showed growth, but they were only grown for 6 days, so we cannot draw any valid conclusions based on qualitative observations, so we will continue to let them grow.

Conclusion

Our results showed that the software we designed is capable of generating usable, and potentially improved, thermometers. While we generated a good deal of useful control data from the plant experiments, the plants did not grow long enough to make any claims about the effectiveness of the enzymes on plant growth.