Part:BBa_K1073034
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 surrounding medium and can be taken up by cells containing the corresponding construct BBa_K1073035.
The expression of aeBlue can be used as selection marker. AeBlue exhibits a dark blue color when expressed and is visible to 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. 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.
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:
Left: 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.
Right: 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal 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 RFC[21]Illegal BglII site found at 2830
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1683
Illegal AgeI site found at 1880 - 1000COMPATIBLE WITH RFC[1000]
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