Team:BCCS-Bristol/Wetlab/signal soil

From 2010.igem.org

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=Signal Visibility in Soil=
=Signal Visibility in Soil=
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==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, mimicing the CCD-based method we envision farmers using to detect fluorescence were this project to be used commercially.
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, mimicing the CCD-based method we envision farmers using to detect fluorescence were this project to be used commercially.
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==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 A<sub>600</sub> value of ~0.5, roughly translating as 2.5x10<sup>9</sup> 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:
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 A<sub>600</sub> value of ~0.5, roughly translating as 2.5x10<sup>9</sup> 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:
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|A sample of the unsterilised soil with a 200μL aliquot of cell culture applied 10 minutes before visualisation
|A sample of the unsterilised soil with a 200μL aliquot of cell culture applied 10 minutes before visualisation
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==Results and Conclusion==
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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.
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.
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Click [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Lab_photos/First_GFP_Experiment here] for some example images from the test.
Click [https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Lab_photos/First_GFP_Experiment 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.
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.

Revision as of 19:04, 25 October 2010



Contents

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, mimicing 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 + 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.