Part:BBa_K515105:Experience

Thermostability

This test is to show the thermostability of sfGFP, by finding the temperature at which the protein denatures. Stock solutions of sfGFP were prepared by extracting the protein from cell lysate, and then 50μl aliquots of the solution were heated in a PCR thermocycler along a temperature gradient.

After two hours, 30μl was removed from each aliquot and diluted with 170μl of 20mM Tris buffer to give 200μl samples. The samples were then measured by fluorescence on a 96-well plate. The corresponding curve was plotted on a graph and the curve was used to calculate the denaturation temperature.

Soil survivability and plasmid retainment testing

Superfolder GFP (sfGFP) can be used as a reporter for the longevity of E. coli cells in soil. We transformed chemically competent DH5alpha cells with the construct and incubated these cells on small filter discs in sterilised and non-sterilised soil. We checked periodically for presence of the GFP-expressing plasmid inside the cells by inoculating the filter discs in LB enriched with antibiotics and growing up bacteria with antibiotic resistance. We recovered fluorescent cells from both sterile and non-sterile soil after six weeks (Figure 1).

Figure 1. Cultures grown from filter discs inoculated with fluorescent E. coli in a) sterile and b) non-sterile soil after six weeks' incubation. c) Negative control comprised of a non-inoculated filter disc placed into soil. Fluorescence is indicated by the orange areas (data by Imperial College iGEM 2011).

Plasmid DNA was extracted using a miniprep kit to analyse the DNA present in the bacteria. A digest with enzymes EcoRI and PstI was used to extract the insert from the vector while another digest with just EcoRI was performed to analyse the size of the open vector (Figure 2).

Figure 2. Gel digest of plasmid DNA from bacteria extracted from sterile and non-sterile soil. The GFP insert is visible just below the 2kb band (data by Imperial College iGEM 2011).

Accordingly, the plasmid was still present in bacteria after six weeks' incubation and was functional as the bacteria exhibited antibiotic resistance as well as fluorescence.

Plant uptake of E. coli

As described by Paungfoo-Lonhienne et al. (2010), plants are able to actively take up microbes. We replicated these findings using E. coli DH5alpha cells expressing sfGFP. Bacteria were grown into exponential phase, spun down and resuspended in 5mM MES to OD 30. 2, 4, and 8ml of these bacteria were added to 100ml half-MS cultures containing three-week old Arabidopsis thaliana Columbia strain wild type plants.

Subsequently, the roots were washed in PBS to avoid imaging of any false positives - bacteria on the outside of rather than inside the roots. The sfGFP allowed us to very clearly identify bacteria inside the roots (Figure 3).

Figure 3. Escherichia coli cells expressing superfolder GFP (sfGFP) can be seen inside an Arabidopsis thaliana root using confocal microscopy after overnight incubation of the plants with bacteria. Roots were washed in PBS prior to imaging to avoid "false positives" of bacteria adhering to the outside of the root (data and imaging by Imperial College iGEM 2011).

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