From 2011.igem.org

Revision as of 14:19, 20 September 2011

Main Results

Expression of indole 3-acetic acid by our bacteria

Salkowski assay

We grew up cultures of our IAA producing E. coli as well as a control culture overnight and the performed a colourimetric assay on the cell filtrate in order to determine if there is a difference in the level of IAA. Our results showed that our Auxin Xpress cells produced more IAA than the control.

Figure 1: Standard curve of Salkowski assay made with synthetic IAA in LB

Figure 2: Cuvettes used to measure OD for the standard curve. As IAA concentration increases, the solution progresses towards red.

Root uptake experiment

We exposed Arabidopsis seedlings to our auxin-producing bacteria to observe their effect on the roots. For this, we used a reporter line that expresses YFP in response to indole 3-acetic acid. As controls, we used a culture of seedlings that was not incubated with any bacteria and a control that was indicated with E. coli not expressing IAA. Fluorescence was brightest in the roots incubated with auxin-expressing bacteria (Video 1).

Video 1. Stack images taken by confocal microscopy converted into three-dimensional videos of Arabidopsis thaliana DR5:3VENUSroots (data and imaging by ICL iGEM 2011).

Uptake of bacteria into plant roots

We observed uptake of GFP-expressing bacteria into plant roots (Fig. 2). The fact that (at least some) plants can actively break up bacteria and use them for nutrients was only reported last year ^[1]. This finding is of extreme importance to our project as the exposure of roots to indole 3-acetic acid will vary significantly if the uptake of the compound takes place inside the roots themselves rather than from the outside. This also presents a novel platform of indirectly engineering plants as delivery of a whole range of compounds will be possible using this method.

Figure 2: 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 "wrong positives" of bacteria adhering to the outside of the root (Data and imaging by Imperial iGEM 2011).

To ensure that the bacteria we imaged were inside the roots, we took stack images that were later converted to three-dimensional views of the roots containing bacteria (Video 2).

Video 2. Confocal stack imaging showing sfGFP-expressing bacteria inside plant roots showing blue autofluorescence (data and imaging by Imperial College iGEM 2011).

E. coli survivability in soil

To assess how well our chassis Escherichia coli is able to survive in soil and for how long a plasmid can be retained, we set up an experiment that consisted of inoculating E. coli into sterile and non-sterile soil. After six weeks of incubation, samples grown up from sterile and non-sterile cultures were still fluorescent.

Figure x. Cultures grown from E. coli inoculated in a) sterile and b) non-sterile soil for the duration of six weeks. c) is a negative control of a culture grown up from non-inoculated soil. Green fluorescence is symbolised by orange areas.

Tracking of bacterial metabolic activity using Dendra2

Dendra2 is a photoconvertable protein (3) that normally exhibits green fluorescence but can be switched, using single photon activation, to fluoresce red. This conversion cannot be reversed. Dendra2 is therefore very useful for observing the metabolism of cells: if all protein in a cell is converted to red fluorescence and it subsequently expresses green fluorescence, the cell has to be metabolically active. As we wanted to know if our cells are metabolically active once they have been taken up into root cells, we performed another plant uptake experiment.

We have made a novel fluorescent protein, Dendra2 available as a BioBrick. Dendra2 normally fluoresces green but can be irreversibly converted to red fluorescence. This is very useful for tracking the viability of cells in different circumstances (for instance, after they have been taken up into root cells). We did a plant uptake experiment with Dendra2-expressing E. coli and converted the protein using a 405nm laser on a confocal microscope (Fig. x).

Figure x. Dendra2 photoconversion in bacteria taken up inside plant roots. 1 is the area photoconverted using the 405nm laser. 2 is an individual bacterium whose Dendra2 protein has undergone photoconversion. 3 is a negative control consisting of a non-photoconverted bacterium. The bacteria found inside the roots can be seen on the right. The data on the left displays the conversion from green to red fluorescence for the highlighted areas. Ch2: emission in green spectrum. Ch3: emission in red spectrum. ChD: brightfield emission.

We also have a pretty nifty time-lapse video of the conversion (Video x):

Video x.

References:

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