"

From 2010.igem.org

Signal Visibility in Soil

Motivation

For this project to work it was essential that a signal could actually be detected when in soil. To test this we mixed E. coli constitutively expressing GFP in soil and imaged and photographed them under a stereo-microscope, mimicking the CCD-based method we envision farmers using to detect fluorescence were this project to be used commercially.

Experiment

Sample soil taken from local source (team member's garden). 6 x ~15g aliquots of soil were placed in 50mL centrifuge tubes, taking care to avoid including wildlife. 3 of the tubes were then sterilised using an autoclave. Meanwhile, a 1 in 10 dilution of the overnight culture of MG1655 cells + [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] was found to have an A₆₀₀ value of ~0.5, roughly translating as 2.5x10⁹ cells/mL in the original culture. The following table lists the rough figures for the 6 soil experiments set up. The lines notated with "S" were for the sterilised tubes, the lines notated with "N" were for the non-sterilised tubes:

S1	~107 cells/g soil	100μL cell culture plus 900μL LB added to soil
S2	~106 cells/g soil	100μL 1/10 dilution of cell culture plus 900μL LB added to soil
S3	~105 cells/g soil	100μL 1/100 dilution of cell culture plus 900μL LB added to soil
N1	~107 cells/g soil	100μL cell culture plus 900μL LB added to soil
N2	~106 cells/g soil	100μL 1/10 dilution of cell culture plus 900μL LB added to soil
N3	~105 cells/g soil	100μL 1/100 dilution of cell culture plus 900μL LB added to soil

The soil cultures were left overnight at 37°C.

Soil samples viewed under fluorescence microscope. Controls used were as follows:

A 200μL aliquot of pure water

A 200μL aliquot of cell culture expressing GFP

A sample of the unsterilised soil

A sample of the unsterilised soil with a 200μL aliquot of cell culture applied 10 minutes before visualisation

Results and Conclusion

All test soil samples showed growth of E.coli expressing GFP. As might have been expected, a significant increase in growth was observed in the sterile samples when compared to the non-sterile samples at similar dilutions of bacteria/g soil, suggesting our lab-safe MG1655s had been out-competed by more robust wild-type bacteria strains.

Click here for some example images from the test.

It quickly became clear that using E. coli spread freely in the soil was not going to yield a strong enough signal to detect from relatively low tech equipment attached to the back of a tractor. It was because of this that we invented the bead method of spreading our bacteria.