Team:SSTi-SZGD/InterLab

SSTi-SZGD---Interlab

Overview

As a new iGEM team, we are pleased to participate in the Fourth International Inter-Laboratory Measurement Study this year.

This year we are required to test six devices either on plate reader or flow cytometry. Our lab conditions only allow us to experiment with plate reader. These six devices contain promoter and a RBS sequence, as well as a reporter gene GFP. The aim of the study was to test the expression level of GFP under different combination of promoter and RBS. Compared to Interlab study 2016, this year there were three additional devices, and each device contains different promoter in association with a newly allocated RBS. In addition, this year each sample was required to be repeated four times, and the observation time for cell reactions were set to be at 0, 2, 4 and 6 hours.

The six devices were tested this year are shown as follows, along with the controls:

Positive control
Negative control
Test Device 1: J23101.BCD2.E0040.B0015
Test Device 2: J23106.BCD2.E0040.B0015
Test Device 3: J23117.BCD2.E0040.B0015
Test Device 4: J23101+I13504
Test Device 5: J23106+I13504
Test Device 6: J23117+I13504

Protocol

OD 600 Reference point

Add 100ul LUDOX 100% into wells A1，A2，A3，A4; add100ul of H2O into wells A2, B2, C2, D2. Measure absorbance at 600nm for all samples in standard measurement modes on plate reader.

Fluorescein fluorescence standard curve

Spin down fluorescein stock tube to make sure pellet is at the bottom of tube. Prepare 2*fluorescein stock solution(100uM) by resuspending fluorescein in 1mL of 1xPBS.
Dilute the 2*fluorescein stock solution with 1xPBS to make a 1*fluorescein solution and the resulted concentration of fluorescein stock solution was 50uM. The plate was set up with the fluorescein stock in column 1 and an equal volume of 1xPBS in columns 2 to 12. A serial dilution was performed by consecutively transferring 100ul from column to column with good mixing.
Then repeated serial dilution for rows B, C, and D. Measured fluorescence in standard measurement modes on plate reader, then imported data into Excel.

Figure 1. Overview of 96-well setup

Cell measurement

Figure 2. illustration of cell measurement workflow

Day 1 :Transformed into Escherichia coil DH5a
Day 2: Picked 2 colonies from each plate and inoculated it into 5-10ml LB medium+Chloramphenicaol. Grew the cells overnight (16-18 hrs) at 37℃and 220 rpm.
Day 3: Cell growth,sampling,and assay. In total four plates were set up to read, one plate was for each time point respectively: 0,2,4,and 6 hours. Each plate was set up as shown below. Measured absorbance at 600 nm and measured fluorescence in standard measurement modes on plate reader, then imported data into Excel.

Figure 3: illustration of cell measurement plate setting

Results

For a more intuitive display of our interlab results, we made the following chart and graphies,by referring to the 2016 interlab formats.

Figure 4: OD600 Reference point, automatically generated by iGEM official forms

Figure 5: A fluorescein standard curve generated by measuring fluorescence of serial dilution

Figure 6: A fluorescein standard curve (in log scale) generated by measuring fluorescence of serial dilution

Figure 7: Absorbance curve measured at OD600 for GFP expression of all tested devices and controls

Figure 8: Fluorescein curve for GFP expression of all tested devices and controls

Figure 9: The fluorescence readings fro each test device or control was corrected for absorbance at OD600

Conclusion

From the results, we suggested that 1) device 2 might be the best device because not only the E. coli. transformants grew normal, but also GFP protein expression was the highest compared to other devices; 2) transformants of devices 1 and 4 did not grow well, but their GFP expression was very high, compared to other devices, therefore we guess that the high protein expression rate somehow slowed down the growth of bacterial cells; 3) transformants of devices 3, 5 and 6 showed normal growth patterns, but their GFP expression levels were comparably low. In conclusion, each device showed different level of GFP expression as well as cell growth condition, which likely to be a direct result of different combination of promoter and RBS sequence.