Team:ZJU-China/Project/Standardization/Results

Standardization: Results

Standardization of Theophylline Riboswitch (BBa_K411003)

We choose BBa_K411003, a typical riboswitch used by many iGEM teams, to be our first riboswitch to be standardized, which was firstly designed by iGEM10_NYMU-Taipei and has been tested by ZJU_China 2012.

Protocol:

1. Cultivate the BL21 containing BBa_K411003 in LB at 37℃180rpm until OD600 reaches 0.5.
2. Induce the bacteria with 1mM IPTG (final concentration), and cultivate them at 37℃180rpm for 4hrs.
3. Dilute the suspension culture with LB to 0.1OD600 . Theophylline is added at concentration ranging from 1.00E-05 mol/L to1.00E-01 mol/L.
4. Measure the RFU ( ex488/em525) and OD600 synchronously.
5. Data Analysis

Fig 1.2 Fig1.2 shows how the whole GFP Change Rate(GCR) changes at different time and under different concentration. Initially, the culture induced by 0.5M Theophylline has the largest whole GCR. And almost every culture’s GCR experiences decrement along time.

Fig 1.3 GFP Change Rate versus Concentration at 100 minutes after Theophylline was added.

2、Standardization of Atrazine Riboswitch (BBa_1054008) Since our project Atrazine Elf is based on the Atrazine-specific Riboswitch , our second choice to be standardized is Atrazine Riboswitch. Protocol: (1).Cultivate the BL21 containing BBa_ in LB at 37℃180rpm until OD600 reaches 0.5. (2).Induce the bacteria with 1mM IPTG (final concentration), and cultivate them at 37℃180rpm for 4hrs. (3).Dilute the suspension culture with LB to 0.1OD600 . Theophylline is added at concentration ranging from 1.00E-06 mol/L to1.00E-01 mol/L. (4).Measure the RFU ( ex488/em525) and OD600 synchronously. (5).Data Analysis

Fig 2.1 Graphic Model about how RFU/OD changes at different time and different concentration of Atrzine.

Fig 2.2 Graphic Model about how GFP Change Rate changes at different time and different concentration of Atrzine.

3、Standardization of Theophylline Riboswitch 2(BBa_1054022 ) Various mechanisms about how the aptamer response to inducer have been discovered and we find out a special riboswitch ,which acts differently from K411003. Protocol: (1).Cultivate the BL21 containing BBa_ in LB at 37℃180rpm until OD600 reaches 0.5. (2).Induce the bacteria with 1mM IPTG (final concentration), and cultivate them at 37℃180rpm for 4hrs. (3).Dilute the suspension culture with LB to 0.1OD600 . Theophylline is added at concentration ranging from 1.00E-06 mol/L to1.00E-01 mol/L. (4).Measure the RFU ( ex488/em525) and OD600 synchronously. (5).Data Analysis

Fig 3.1 Graphic Model about how RFU/OD changes at different time and different concentration of Theophylline.

Fig 3.2 Graphic Model about how GFP Change Rate changes at different time and different concentration of Theophylline.

4、Data Analysis Theoretically, data processing should consist of two stages including Processing and Calibration, respectively. Lacking enough time and equipment, we cannot finish the Calibration part-which is about to find out the relation between OD600 and the number of cells and also the relation between RFU and mass of GFP-before wiki freeze ,which may have some influence on our results. Here comes the Processing Part: (1).Subtract the backgrounds from the raw data A_corrected=A_raw-A_background G_corrected=G_raw-G_background (2).Calculate the RFU of per unit, P P=G_corrected/A_corrected (3).Calculate the whole RFU change rate, S S=(dG_corrected)/dt (4).Plot

If given more time, we may be able to measure the relation between OD and the number of cells AND RFU and mass of GFP, and thus we can calculate the PoPS, which is more accurate and suitable to describe the action of engineered devices.

Limited by our time and resources, it is impossible for us to standardize all the riboswitches. Any teams or individuals are warmly welcomed to enrich our riboswitch standardization library.