Difference between revisions of "Part:BBa K1073034"

Revision as of 15:05, 1 October 2013

Inducible ampR expression combined with LasR, RhlI and aeBlue expression cassettes

This device 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 las quorum sensing system N-buturyl 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

This device is intended to be used in concert with BBa_K1073035. 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.

Possible Applications

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_K1073034 when synthetic N-3-oxododecanoyl-HSL is added to the culture broth.

Growth Characteristics

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 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 and placed on top of the agar plate. Growth could only be observed around the filter paper due to Diffusion of N-3-oxododecanoyl in the the agar.

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: 1 µM N-3-oxododecanoyl-HSL, 10 µM N-3-oxododecanoyl-HSL, 100 µM N-3-oxododecanoyl-HSL and negative control (2xYT medium).

Production of rhl autoinducer N-buturyl-HSL

Expression of chromoprotein aeBlue

iGEM Team Braunschweig 2013: Expression of chromoproteins expression cassettes amilGFP (BBa_K1073024), eforRed (BBa_K1073022) and aeBlue (BBa_K1073020) in E. coli. The aeBlue expression cassette is included in BBa_K1073034 and cells containing this construct can be easily identified by this marker.

Sequence and Features