(241a) Resumption of Bioluminescent Gene Expression in Whole-Cell Bacterial Biosensors After Performing High Temperature Switch

According to Frost and Sullivan, the global market for biosensors is projected to grow from $5.11 billion in 2006 to $10.62 billion in 2013 as biosensors have emerged as a leading technology in the application of analyte detection and analysis. Whole cell biosensors, which use living microbial organisms to detect changes in their environment, have been shown to exhibit the potential to both detect the presence of targeted chemical substances with high selectivity and determine their concentrations with high sensitivity (in qualitative and quantitative sensing properties).[1] The Pseudomonas putida strain TVA8 bacterial-based biosensor is used to sense parts-per-billion levels of chemicals or inducers (i.e. trichloroethylene, TCE) through bioluminescence. However, the technical challenge in detecting changes in inducer concentrations is to engineer an accurate quantitative link between the bioluminescence signal and the inducer concentration.

It has been shown previously that the bioluminescence signal can be ?turned off? or ?reset? utilizing temperature.[2] It would be beneficial to continuously ?turn-off? the bioluminescence signal so that the signal from the biosensor can be ?re-set? after receiving a measurable response of TCE concentration for calibrating the bioluminescence signal. Previously discovered was a method for ?turning off? the bioluminescence in P. putida strain TVA8, through controlled heating to denature proteins involved in the bioluminescence mechanism. The bacteria were grown at 27 °C on TCE-saturated minimal media shaking at 220 RPM. At 8 hours, the temperature of the bacteria was increased to 37 °C using a water bath. The bioluminescence was recorded once per hour prior to the temperature change and once per minute after the change. After raising the temperature, the bioluminescence signal was found to decrease rapidly, with an average of 95.388 ± 1.59% of the maximum bioluminescence eliminated after 2 minutes. Therefore, it is possible to eliminate the bioluminescence signal using a high temperature (37 °C) control ?switch?, which is necessary for sensor calibration.

The goal of this study is to verify this high temperature control ?switch? can be used to calibrate the bioluminescence signal. Additionally, resumption of the bioluminescence signal is tested after the high temperature (37 °C) control ?switch? is performed for calibration purposes as well. The bacteria were grown at 27 °C on TCE-saturated minimal media for 7 hours. After the 7th hour, the high temperature (37 °C) control ?switch? was performed for approximately 30 minutes causing the bioluminescence signal to ?turn off? after 15 minutes. Afterwards, the temperature of the bacteria is decreased back to 27 °C and after 30 minutes the bioluminescence signal resumed. Resumption of the bioluminescence signal shows that the signal can be calibrated in order to read multiple events in succession.

[1] Applegate, B.M., et al. Applied and Environmental Microbiology, Vol. 64, No.12, 1998.

[2] Callahan, D., et al., ?Inhibition of bioluminescent gene expression in whole-cell bacterial biosensors using a high temperature switch,? AIChE National Conference, San Francisco, CA, 2006.