Difference between revisions of "Part:BBa K1073035"

Revision as of 18:25, 2 October 2013

Inducible ampR expression combined with RhlR, LasI and eforRed expression cassettes

This device is a combination of BBa_K1073028 and BBa_K1073032 and includes parts of the rhl and las quorum sensing system of Pseudomonas aeruginosa. It includes constitutive expression cassettes for RhlR, LasI and eforRed.

The expression of ampR is regulated by the promoter of the rhl system. In order to induce ampR expression the transcription regulator RhlR and the specific autoinducer molecule N-buturyl homoserine lactone have to be present. N-buturyl-HSL is synthesized by RhlI (this protein is not included in the construct). However, N-buturyl-HSL can be added to the culture broth. RhlR and N-buturyl-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].

LasI synthesizes the specific autoinducer of the las quorum sensing system N-3-oxododecanoyl homoserine lactone. The autoinducer molecules are secreted into the medium.

The expression of eforRed can be used as selection marker. EforRed exhibits a strong pink 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_K1073034. 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_K1073035 and BBa_K1073034 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-buturyl-HSL is added to the culture broth.

Growth Characteristics

iGEM Team Braunschweig 2013: Growth curve of E. coli Top10F containing BBa_K1073035 on the high copy plamsid pSB1C3. Comparison of growth in ampicillin containing complex medium in presence and absence of rhl autoinducer N-buturyl-HSL.

Induction of ampR expression by N-buturyl-HSL

Growth of cells containing BBa_K1073035 can be induced by synthetic autoinducer molecules N-buturyl-HSL added to the medium containing ampicillin. A piece of filter paper was soaked with N-buturyl-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-buturyl into the solid medium. The amount of colonies and the radius of growing cells depend on the concentration of the autoinducer.

Production of las autoinducer N-3-dodecanoyl-HSL

Expression of chromoprotein aeBlue

This device encodes the eforRed expression cassette (see BBa_K1073022). When expressed this chromoprotein exhibits a pink color and therefore can be used as a selection marker for cells containing BBa_K1073035. 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 eforRed 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.

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iGEM Team Braunschweig 2013:

Left: Cell pellet and liquid culture (2xYT medium) of cells containing BBa_K1073035. Cells turn red due to the constitutive expression of the chromoprotein eforRed encoded in this device.

Right: Absorption spectrum of chromoprotein eforRed in a wavelength range of 400-800 nm.

Sequence and Features