Team:NTHU Taiwan/Experiments
Experiment
Biobrick designWe referred to DNA sequences of Fac-dex gene in Burkholderia sp. FA1 and Pseudomonas fluorescens. We inserted Fac-dex gene into plasmid backbone pSB1C3 for protein expression and transform this modified plasmid into E.coli strain, BL21.
In order to measure Fac-dex expression, we add GFP behind Fac-dex. By fluorescence microscope, we can detect GFP fluorescence to know the expression of Fac-dex .
We use Lacl promoter to control protein expression.
Experimental design
We culture E.coli in liquid medium containing PFOA with different concentration.
We worked on protein expression test via SDS-PAGE and GFP fusion.
Last but not least, we trace the degradation by getting the concentration of fluoride via ion chromatography. As long as knowing the concentration of fluoride, we can estimate the amount of PFOA consumed by engineered bacteria.
We added sodium fluoride solution of different concentration into liquid medium.
We worked on protein expression test of CRCB via SDS-PAGE and GFP fusion.
Last but not least, we test the function of CRCB by incubating E.coli in the medium containing over 0.08M sodium fluoride.
We get waste water sample from waste water treatment plant and we filtered the waste water sample to get the water without other living things; that is, we test the growth of E.coli under chemical condition of waste water.
Detection of FluorideTo measure the concentration of fluoride, we applied both ion chromatography system and fluorine ion selective electrode to assure the accuracy of data. After knowing the concentration of fluoride, we estimated the amount of PFOA degraded by engineered bacteria through reaction mechanism. Following is brief introduction to this method.
Ion chromatography relies on the attraction between oppositely charged stationary phase, known as an ion exchanger, and analyte. The ion exchanger consists of an inert support medium coupled covalently to positive (anion exchanger) or negative (cation exchanger) functional groups. To these covalently bound functional groups, the oppositely charged ions are bounded (mobile counter ion), which will be exchanged with like charge ions in the sample having charge magnitude more than the ions bounded to the matrix. Thus if anion exchange chromatography is performed, negatively charged sample components will interact more with the stationary phase and will be exchanged for like charged ions already bounded to the matrix.
An ion selective electrode (ISE) is a sensor which converts the activity of a specific ion dissolved in a solution into an electrical potential which can be measured by a voltmeter or PH-meter. A fluoride selective electrode is a type of ion selective electrode sensitive to the concentration of the fluoride ion. A common example is the lanthanum fluoride electrode. In the lanthanum fluoride electrode, the sensing element is a crystal of lanthanum fluoride LaF3, doped with europium fluoride EuF2 to create lattice vacancies. Such a crystal is an ionic conductor by virtue of the mobility of fluoride ions which jump between lattice vacancies. As transference of charge through the crystal is almost exclusively due to fluoride, the electrode is highly specific to fluoride. The only ion which significantly interferes is hydroxide (OH−). Generally such "alkaline error" can be avoided by buffering the sample to a pH below 7.
