Team:WashU StLouis/InterLab

As a team, we decided to participate in iGEM's Fifth International InterLab Study along with other teams from around the world. This study is organized by iGEM's measurement committee in an effort to better measurement techniques in the field of synthetic biology. Our involvement in the study required that we submit measurement data dealing with the fluorescence and absorbance associated with cells transformed with different test devices. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices), and we measured the absorbance and fluorescence of our samples using a Tecan Infinite M200 Pro plate reader. We divided the protocol into six components: (1) measuring the OD600 reference point of LUDOX CL-X, (2) construction of a particle standard curve, (3) construction of a standard fluorescence curve, (4) transforming the two controls and 6 test devices into competent DH5α cells, (5) conducting Abs600 and fluorescence measurements of transformed DH5α cells, and (6) calibration of OD600 into colony forming unit (CFU) counts which are directly related to viable cell counts.

Step One: Measurement of LUDOX CL-X OD600 Reference Point

We used LUDOX CL-X as a reference to obtain a conversion factor to determine an OD600, similar to that acquired from a spectrophotometer, from an Abs600 read from the plate reader.

Abs600 and OD600 Data/Calculations for LUDOX CL-X and Water

Step Two: Construction of Particle Standard Curve

We measured the Abs600 of a dilution series of a known concentration of silica microspheres, which mimic the size and optical characteristics of cells, and constructed a particle standard curve. This enabled us to convert Abs600 values into an estimated number of cells.

Abs600 Readings from Silica Microspheres Dilutions

Calculated Particle Counts from Measured Abs600

Step Three: Construction of Standard Fluorescence Curve

We used a dilution series of fluorescein, a molecule that shares similar excitation and emission properties with GFP, to create a standard curve of fluorescence for fluorescein concentration and provide a method of comparison between teams for GFP expression in the transformed E. coli cells.

Fluorescence Measurements from Fluorescein Dilutions

Calculated Correspondence between Measured Fluorescence and Fluorescein Concentration

Step Four: Transformation of Test Devices and Controls

We transformed all of the control plasmids and test device plasmids into E. coli DH5α cells. After transforming the cells successfully and growing them on plates overnight, we picked two colonies from each plate and prepared overnight cultures for them.

Plated DH5α cells after transformation with both high and low concentration of cells

Step Five: Cell Measurements

After growing overnight cultures of our transformed cells, we took both Abs600 and OD600 measurements of our cells. Using the constructed particle standard curve and fluorescence curve, we were able to derive fluorescence per OD and fluorescence per particle values.

Raw Abs600 and OD600 readings from cells

Calculated fluorescence per OD

Calculated fluorescence per cell

Step Six: Calibration of OD600 into Colony Forming Units (CFU)

We grew new plates of transformed DH5α with both the positive and negative controls and began two overnight cultures from each transformation.

Plated DH5α cells after transformation with both high and low concentration of cells

We diluted the sample to yield a 0.1 OD600 using calculations from the measured OD600 of a 1:8 dilution of our overnight cultures.

Measured Abs600 and calculated OD600 and volumes for dilution

For Positive Control Sample 1, we added 25 µL of culture to 975 µL media to make a total volume of 1000 µL.

For Positive Control Sample 2, we added 28 µL of culture to 972 µL media to make a total volume of 1000 µL.

For Negative Control Sample 1, we added 21 µL of culture to 979 µL media to make a total volume of 1000 µL.

For Positive Control Sample 2, we added 23 µL of culture to 977 µL media to make a total volume of 1000 µL.

Measured Abs600 and calculated OD600 values

Next, we performed a dilution series with triplicates for each overnight culture and plated the 8*10-3, 8*10-4, and 8*10-5 dilutions. Assuming that one bacterial cell gives rise to one colony, we counted the number of colonies per plate and multiplied by the final dilution factor to obtain a CFU/mL value for a starting sample of OD600.

Plated DH5α cells of different dilution factors from culture of transformed cells

Measured number of colonies and CFU/mL in a Starting Sample of OD600 = 0.1