Part:BBa_K515105

J23100 promoter - sfGFP

A composite part between J23100 and superfolder GFP (sfGFP).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 215
  
• 1000
  COMPATIBLE WITH RFC[1000]

Background

This part is superfolder GFP, a very brightly fluorescent protein under the control of the constitutive promoter J23100. This BioBrick has been sequence verified.

Thermostability

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

After two hours, 30 μl was removed from each aliquot and diluted with 170 μl of 20 mM Tris buffer to give 200 μl 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.

Results of the heat denaturation experiment. The temperature at which half the proteins are denatured was studied by measuring fluorescence (PTm50) mRFP1: 82.2°C; GFPmut3b: 61.6°C; Dendra2: 89.1°C; sfGFP: 75.0°C.

The sigmoidal curves that were calculated gave us the following function in order to find K which we also call PTm50 (temperature at which half of the proteins are denatured studied by measuring fluorescence):

Soil survivability and plasmid retainment testing

To test for the survivability of E. coli in soil, we set up an experiment. We initially transformed chemically competent DH5alpha cells with superfolder GFP. These cells were inoculated on small (about 0.5 cm diameter) filter discs, which were placed in autoclaved and non-autoclaved soil. We periodically grew up cultures from these filter discs over the course of six weeks.

After six weeks, we were able to recover fluorescent bacteria from sterilised soil. Colonies appearing to be on the plates from non-sterile soil had lost fluorescence (Fig. 2).

Figure 2. Colonies recovered from filter discs and grown on LB plates containing selective antibiotics imaged using a LAS-3000 gel imager. a) Sample taken from non-sterilised soil b) Sample taken from sterilised soil. (Data by Imperial College London iGEM team 2011).

As is visible from these plates, fluorescence was detected in bacteria recovered from both sterile but not from non-sterile soil. The control plate showed that there was no contamination with other fluorescent lab bacteria. In order to investigate whether the fluorescence observed was due to the presence of the original sfGFP construct and whether the E. coli-like colonies from the non-sterile sample had retained a plasmid we extracted plasmid DNA using a miniprep kit and did a digest with EcoRI and PstI and with EcoRI on its own to check for presence of the original insert and size of the unfolded vector, respectively (Fig. 3).

Figure 3. Gel digests of bacteria displaying colony morphology typical of E. coli recovered from non-sterilised and sterilised soil. These bacteria exhibited colony morphologies typical of E. coli. (Data by Imperial College iGEM team 2011)

The insert is very clearly visible at just below 2 kb. This confirms the presence of superfolder GFP in both cultures. Sequencing of the GFP insert revealed that a single frameshift mutation had taken place in the colonies grown up from non-sterile soil. No mutations were observed in the superfolder GFP gene contained in the bacteria inoculated in non-sterile soil. This explains the absence and presence of fluorescence in the respective colonies. However, the bacteria were still resistant to the antibiotics and contained the plasmid. We will be replicating these results with other samples to ensure this is representative.

In addition, small colonies appeared on the non-sterile plate that had very different colony morphology. We grew this colony up in LB medium containing selective antibiotic and subsequently performed a separate miniprep. No DNA was yielded in this miniprep. It is therefore likely that the plasmid was not transferred to these bacteria but that they either possess natural antibiotic resistance or were able to survive on plates that whose antibiotics had already been depleted by the presence of resistant engineered bacteria.

Plant uptake of E. coli

As described by Paungfoo-Lonhienne et al. (2010)^[1], 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 8 ml of these bacteria were added to 100 ml 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).

References:

[1] Paungfoo-Lonhienne, C. et al. (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS One, 5(7), e11915.