Part:BBa_K515107
Composite p(tetR) - Dendra2
Description
This BioBrick comprises the coding sequence BBa_K515007 under the control of the repressible promoter TetR BBa_R0040 with the RBS BBa_B0034. The coding sequence BBa_K515007 codes for Dendra2, a photoconvertible reporter protein. In its natural state, it is excited and emits at 486 and 505 nm, respectively. This appears green and therefore in its natural state Dendra2 can be used as a GFP reporter. However it can be irreversibly converted to be excited at 558 nm and consequently emit at 575 nm. After conversion reporter appears red and can be observed as RFP. Conversion can be efficiently achieved at wavelengths of both 488 and 405 nm [1]. However, the cytotoxic wavelength of 405 nm converts Dendra2 more efficiently (data not shown).
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
This BioBrick has been sequence verified.
Characterisation
This part (BBa_K515107) has been characterised in a number of aspects to test its properties as a reporter. The tests describe this part in terms of thermostability, photostability and photoconversion.
Thermostability
This test is to show the thermostability of Dendra2, by finding the temperature at which the protein denatures. Stock solutions of Dendra2 were prepared by extracting the protein from cell lysate, and then 50 μl aliquots of the solution were heated in a PCR thermocycler along a 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 fluorescence of the samples was measured on a 96-well plate. The corresponding curve was plotted on a graph (Figure 1).
Figure 1: Results of the heat denaturation experiment. The temperature at which half of the protein is denatured measured by looking at its fluorescence (PTm50) mRFP1: 82.2°C; GFPmut3b: 61.6°C; Dendra2: 89.1°C; sfGFP: 75.0°C (click on graph to enlarge).
Photostability
This test is to show photostability of the Dendra2 protein for green fluorescence without the conversion to red fluorescence. Green (505 nm wavelength) and red (575 nm wavelength) fluorescence emission was measured for Dendra2-expressing cells over time. As control RFP and GFP expressing cells have been used to compare the green and red fluorescence emission. Red and green fluorescence of control LB medium was also measured as a blank that can be subtracted from the readings of the GFP, RFP and Dendra2 expressing cells.
Photoconversion
Fig. 2:Graph showing the photostability of Dendra2. The excitation wavelength of 486 nm does not cause photoconversion even though the wavelength of 488 nm does (click on graph to enlarge).
Purified Dendra2 protein was measured for the red fluorescence emission after photoconversion using single photon stimulation at 405 nm wavelength. It was exposed to the RFP excitation wavelength (558 nm) and at a set time point it was also exposed to 405 nm photoconversion wavelength.
Figure 3: Red flourescence emission of Dendra2 protein upon single photon stimulation at 405 nm wavelength. Red flourescence is very low before photoconversion, however at time point 25 s after the start of the measurement 405 nm photoconverting wavelength was applied. An increase in red flourescence emission can be observed between time point 25 s and 319 s, after which red flourescence emission levels off.
This part has been used as a reporter for observation of bacterial uptake into the roots of the plants. Due to its photoconvertible properties, it allows monitoring of the metabolic activity of bacterial cell once uptaken into the root. Dendra2 was converted from 486 nm excitation and 505 nm emission wavelength, to 558 nm excitation and 575 nm emission wavelength using single photon stimulation. Conversion was achieved after exposure to 405 nm wavelength using laser. Photoconversion was completed after about 15 rounds of bleaching at 50% laser intensity with the pinhole set to 3 airy units.
Figure 4. Dendra2 photoconversion in bacteria taken up inside plant roots. The graph on the top displays averaged fluorescence over the entire photoconverted area and the amount of brightfield light (the background light used to see the outline of the roots) recorded is therefore very high. The graph on the bottom displays emission at green and red fluorescence over the same time span. |
Video 1. This video shows the photoconversion of Dendra2 within E. coli cells that have been taken up into the plant roots as a time-lapse of pictures taken after each round of bleaching at 405 nm. The targeted area of cells being photoconverted corresponds to the top graph in Figure 4. There is a single bacterium visible on the right that was not targeted for photoconversion and serves as a control (data and imaging by Imperial College iGEM 2011). |
Intensity ROI 1
After exposure with 405 nm wavelength, the cells are observed to steadily decrease fluorescing in green emission spectra over a time period of 140 seconds. In the same time frame the cells are observed to increase fluorescing in the red spectra, with fluorescence in the two emission spectra being equally intense at 20 seconds after the photoconversion. Brightfield emmision is kept at just over 100 units throughout the duration of observation of photoconversion. Brightfield is present for visualisation of the root and bacterial cells without flourescence.
Intensity ROI 2
ROI 2 displays the fluorescence emitted by a single cell while it is being converted from green to red emission. Emission of green spectra fluorescence can be observed to decrease steadily over time period of 140 seconds. In the same time frame the cell can be observed to fluoresce more strongly in the red spectra. The difference between red and green emission is greater in ROI 2 than in ROI 1 due to single cell focus eliminating backround emission which can be seen in ROI 1 that causes the difference between two spectra in ROI 1 to be smaller than in ROI 2. Brightfield emmision is kept at just over 100 units throughout the duration of observation of photoconversion. This is necessary for visual observation of cells within the root.
A single bacterium on the right (visible in the video above) is used as a negative control. It was not targeted for bleaching by the laser and can be observed to remain green through the conversion process.
Method
The bacterial cells within root were visualised using a Zeiss LSM-510 inverted confocal microscope. The induction of photoconversion was performed using a laser at 405 nm wavelength. The bacterial root uptake experiment used the same protocol as previously described by Paungfoo-Lonhienne et al. [2]Using Dendra2 to trace metabolic activity
To assess whether Dendra2 could be used to track the metabolic activity of E. coli, culture of Dendra2 expressing E. coli were photoconverted with a blue laser and measured fluorescence during incubation at 30°C to see if green fluoresence re-emerged. As expected, green fluorescence decreased after photoconversion and red fluorescence increased. As the sample was left to incubate at 30˚C, green fluorescence re-emerged as the bacteria are producing more Dendra2. This proves that the cells remain metabolically active and that metabolic activity can be tracked using Dendra2. Surprisingly red fluorescence also increased over time without additional photoconversion. We suspect that this may be a delayed response from the initial photoconversion.
Figure 36: Green and red fluorescence emitted by E. coli expressing Dendra2, before and after photoconversion with a blue laser.
Figure 37: Re-emergence of green fluorescence over time as cells produce new Dendra2 protein that has not been photoconverted.
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To assess whether E. coli remain metabolically active inside Arabidopsis roots, Dendra2 was photoconverted inside Arabidopsis roots and left in cygel over night so that they could be re-imaged after 24 hours. The roots were imaged four days after infection with Dendra2 expressing E. coli. The bacteria were shown to continue expressing green Dendra2 protein, proving that they are metabolically active inside the roots (Video 7).
Video 7. Photoconversion of Dendra2 in Arabidopsis thaliana roots. After 24 hours, the bacteria were re-imaged at the same settings. Expression of green Dendra2 had increased, showing that the bacteria are metabolically active inside the roots (Imaging by Mark Scott for Imperial College iGEM 2011).
ImageJ was used to analyse the relative fluorescence intensities in the different samples. By determining the percentage change in green fluorescence after photoconversion and 24 hours incubation in relation to the initial level of green fluorescence (figure 36), we can clearly see that green fluorescence decreased dramatically after photoconversion (as expected by the properties of Dendra2). Additionally, after 24 hours the initial green fluorescence level was re-established and slightly surpassed due to production of new Dendra2 protein by the metabolically active bacteria. No red fluorescence was re-converted to green fluorescence because Dendra2 irreversibly changes from fluorescing green to red. There was a high background fluorescence in the red spectrum as roots are highly autofluorescent in the same spectrum.
This result is significant in that we have proven that E. coli remain metabolically active inside the roots for up to 5 days which would allow IAA production by our engineered bacteria once inside the root, thereby enhancing root growth.
The use of other reporters such as GFP would be sufficient to demonstrate whether our bacteria have localised inside the root or not but not to show that they are metabolically active because it could just be residual green fluorescence.
Figure 38: Relative fluorescence of Dendra2-expressing bacteria inside Arabidopsis roots. The bacterially infected roots were imaged on the confocal microscope, photoconverted and imaged again, and left overnight to be imaged after 24 hours. This graph shows the percentage change of green fluorescence after photoconversion and 24 hours incubation in relation to the initial green fluorescence.
References:
[1] Gurskaya N et al. (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nature Biotechnology 24: 461-465.
[2] Paungfoo-Lonhienne et al. (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS ONE 5(7):, e11915. http://www.nih.gov/science/models/arabidopsis/index.html
| chassis | E. coli DH5α |
| control | R0040 |
| device_type | Fluorescent reporter |
| emission | 505 nm and 575 nm |
| excitation | 486 nm and 558 nm |
| protein | K515007 |
| rbs | B0034 |
| resistance | Chloramphenicol |