Team:BOKU-Vienna/Measurement
Measurement
An important aspect of our iGEM BOKU Vienna Team’s new diagnostic method for Buruli Ulcer is the simple and clear visual readout that does not require complex equipment or training. We developed a riboswitch-based readout that can be interpreted with the naked eye. The riboswitch consists of an aptamer-based induction of T7- Polymerase and amilCP under the control of the T7- Promoter.
Our motivation is to apply this method to the neglected tropical disease Buruli Ulcer, however, simply by switching out the aptamer sequence to a different target and designing intrinsic termination, our project can be applied to other diseases or to screen for toxins in the environment.
In order to achieve outstanding results, we have not only performed qualitative but also quantitative measurements.
The qualitative measurements are based on the expression of Blue Chromoprotein amilCP for a positive signal due to the binding of an inducer to the aptamer, which can be easily seen with the naked eye. Our team took pictures of Escherichia Coli DH10B strain induced under different concentrations of Theophylline and different incubation time. Our theoretical model predicted the production of Blue Chromoprotein to be seen after 45 minutes. Therefore, we wanted to perform half hour- to hourly measurements of the construct. For the detailed experimental work see
“Lab Journal”, for data and analysis see
“Results” and
“Demonstrate”.
For quantitative measurements we concentrated on fluorescence data from in vivo and cell-free experiments as well as optical density data from in vivo experiments. GFP fluorescence was measured in a “Tecan Infinite 200” plate reader using the fluorescein standards from the measurement kit in order to control and determine the correct working procedure of our experimental protocols. The data with GFP was converted from arbitrary units to comparable units for an independent standard. Optical density was measured with Hitachi Photometer U-1900 at 600nm to normalize the data to OD600 = 1.0. It is very important to keep the effective range of measurement of the photometer in mind. Samples have to be diluted accordingly to the linear range, which in our case was OD600 of 0.1 to 0.7.
Cell-free expression of our gene of interest with BBa_K3015002 was measured for two reasons:
Working with synthetic-mycolactone, a non-commercially available toxin, came with safety regulations and waste management. Using micro reaction tubes with low reaction volumes is easier than using glass eprouvettes with higher doses of the toxin. Also, diffusion into the cell as well as other possible interactions between the toxin and living cells do not play a role in cell-free expression systems. Measuring GFP was done the same way as explained in the in vivo experiments. The only difference was the low reaction volume of the myTXTL Sigma 70 master mix (sponsored by Arbor Biosciences) of 13µL, which was simply diluted with 1xPBS buffer to a total volume of 50µL per well.
Measurements – GFP Fluorescence
In order to make the riboswitch screening easier, we wanted to provide comparable numbers about the leakiness of the uninduced module by testing the riboswitch with GFP as a reporter gene. By using the fluorescein standards from the measurement kit, fluorescence values can be converted into comparable units. Figure 1 shows the power function of the fluorescein standard curve (log scale). Net mean fluorescence values can be put into the power function as y-values to calculate the corresponding µmol/L fluorescence molecules.
For example: the Theophylline riboswitch with an uninduced net mean fluorescence of 1558.66 equals 0.07526 µM GFP-molecules. Through the OD600 value the number of cells per mL can be calculated. For Escherichia coli with an OD600 of 1.0 = 8*108 cells per mL. To measure reliable OD600 values, samples must be measured in the linear range of the photometer and therefore often need to be diluted and the real OD600 calculated. With anAn OD600=3.81 the number of cells per mL equals 3,048,000,000. The concentration of fluorescent molecules divided by the number of cells per mL leads to 2.47 * 10-20 mol per cell. Which are 15,000 molecules per cell. The activity and increase in gene expression after induction can be measured without standards. The net mean fluorescence values can be normalized to OD600=1.0 by dividing each fluorescence value through the corresponding OD600 value. The normalized fluorescent values can be divided through the uninduced fluorescence to obtain the relative expression or fold increase (see figure 2). Figure 2 shows the fold increase at different inducer concentrations. The concentrations of 0 µM, 1 µM, 10 µM, 100 µM and 1 mM of Theophylline were used for induction. As presented in Figure 2, the Theophylline riboswitch shows low response to 1µM Theophylline induction, a 1.5 fold increase in the presence of 10µM Theophylline, a 4 fold increase of gene expression at 100µM Theophylline and a 6.4 fold increase at 1mM Theophylline.
Our system can work with many different riboswitches. The measurements for the leakiness threshold of our cassette of around 15,000 molecules per cell can be used in the design of other systems. It can be achieved with many riboswitches by simply changing the promoter to reach the desired expression strength.
