Inducible ampR expression combined with LasR, RhlI and aeBlue expression cassettes This device is intended to be used in concert with BBa_K1073035. Using both BBa_K1073034 and BBa_K1073035 a synthetic consortia between two strains can be created when they are incubated in ampicillin containing medium. The two strains depend on each other and can only grow when the other is present in the culture broth.
BBa_K1073034 is a combination of BBa_K1073030 and BBa_K1073033 and includes parts of the rhl and las quorum sensing system of Pseudomonas aeruginosa. It includes constitutive expression cassettes for LasR, RhlI and aeBlue.
The expression of ampR is regulated by the promoter of the rhl system. In order to induce ampR expression the transcription regulator LasR and the specific autoinducer molecule N-3-oxododecanoyl homoserine lactone have to be present. N-3-oxododecanoyl-HSL is synthesized by LasI (this protein is not included in the construct). However, N-3-oxododecanoyl-HSL can be added to the culture broth. LasR and N-3-oxododecanoyl-HSL build a complex that binds to specific sequences of the promoter region and induces expression of the downstream gene (in this case ampR) [Medina et al., 2003].
RhlI synthesizes the specific autoinducer of the rhl quorum sensing system N-butyryl homoserine lactone. The autoinducer molecules are secreted into the medium and can be taken up by cells containing the corresponding construct BBa_K1073035.
The expression of aeBlue can be used as reporter to identify cells bearing this construct. AeBlue exhibits a dark blue color when expressed and is visible to the naked eye in less than 24 h during incubation on agar plates or in liquid culture.
Usage and Biology
Two different strains containing BBa_K1073034 and BBa_K1073035 respectively will together synthesize all molecules required to induce the promoters of the rhl and las system and thus activating the expression of ampR of one another.
Using both BBa_K1073034 and BBa_K1073035 a synthetic consortia between two strains can be created when they are incubated in ampicillin containing medium. The two strains depend on each other and can only grow when the other is present in the culture broth.
It can also be used without BBa_K1073035 when synthetic N-3-oxododecanoyl-HSL is added to the culture broth.
This device was tested in E. coli JM109. During batch cultivation in complex medium containing ampicillin growth of cells is inhibited unless autoinducer molecules (N-3-oxododecanoyl-HSL) are added. Note that due to the leakiness of the promoter a low concentration of clavulanic acid was added to the culture broth to inhibite the background activity of the beta lactamase. Clavulanic acid itself is a beta lactam and acts as a beta lactamase inhibitor. It covalentely binds to the active site and irreversible inactivates the beta lactamase [Fisher et al., 1978].
iGEM Team Braunschweig 2013: Growth curve of E. coli JM109 containing BBa_K1073034 on the high copy plamsid pSB1C3. Comparison of growth in ampicillin containing complex medium in presence and absence of las autoinducer N-3-oxododecanoyl-HSL.
Induction of ampR expression by N-3-oxododecanoyl-HSL
Growth of cells containing BBa_K1073034 can be induced by sythetic autoinducer molecules N-3-oxododecanoyl-HSL added to the medium containing ampicillin. A piece of filter paper was soaked with N-3-oxododecanoyl-HSL solution of different concentrations and placed on top of the agar plate. Growth could only be observed around the filter paper due to the diffusion of N-3-oxododecanoyl into the solid medium. The amount of colonies and the radius of growing cells depend on the concentration of the autoinducer.
iGEM Team Braunschweig 2013: Induction of growth of cells containing BBa_K1073034 on 2xYT agar plates containing ampicillin (0.29 mM) by synthetic autoinducer N-3-oxododecanoyl-HSL of different concentrations.
From left to right: Negative control (2xYT medium), 1 µM N-3-oxododecanoyl-HSL, 10 µM N-3-oxododecanoyl-HSL and 100 µM N-3-oxododecanoyl-HSL.
The same phenomenon was observed when the filter paper was soak with sterile supernatant of a cell culture containing BBa_K1073035. In this case the supernatant contains N-3-oxododecanoyl-HSL produced by LasI encoded in BBa_K1073035 (see experience page).
Production of rhl autoinducer N-butyryl-HSL
In order to verify the production of rhl autoinducer N-butyryl-HSL by the autoinducer synthase RhlI encoded in this device a E. coli JM109 bioluminescence reporter strain was used which contains a rhlRI'::luxCDABE cassette on a pSB406 plasmid backbone [Winson et al., 1998]. The presence of N-butyryl-HSL in the medium leads to the expression of luxCDABE . E. coli JM109::rhlRI'::luxCDABE was incubated with diluted supernatant of a culture of cells containing BBa_1073034. Bioluminescence was measured by using a plate reader.
iGEM Team Braunschweig 2013: Measurement of bioluminescence produced by reporter strain E. coli JM109::rhlRI'::luxCDABE due to the presence of N-butyryl-HSL produced by RhlI in the medium.
Expression of chromoprotein aeBlue
This device encodes the aeBlue expression cassette (see BBa_K1073020). When expressed this chromoprotein exhibits a dark blue color and therefore can be used as a selection marker for cells containing BBa_K1073034. The color is visible to naked eye in less than 24 h on agar plate and in liquid culture.
In order to avoid absorption by aeBlue during OD measurement of a bacterial culture the absorption spectrum was measured. An optimal wavelength for spectral optical density measurement was determined to be at 520nm according to this spectrum.
iGEM Team Braunschweig 2013:
Left: Cell pellet and liquid culture (2xYT medium) of cells containing BBa_K1073034. Cells turn blue due to the constitutive expression of the chromoprotein aeBlue encoded in this device.
Right: Absorption spectrum of chromoprotein aeBlue in a wavelength range of 400-800 nm.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12INCOMPATIBLE WITH RFCIllegal NheI site found at 1306
Illegal NheI site found at 1329
Illegal NheI site found at 2988
Illegal NheI site found at 3011
- 21INCOMPATIBLE WITH RFCIllegal BglII site found at 2830
- 23COMPATIBLE WITH RFC
- 25INCOMPATIBLE WITH RFCIllegal NgoMIV site found at 1683
Illegal AgeI site found at 1880
- 1000COMPATIBLE WITH RFC