Part:BBa_K1073035
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.
iGEM Team Braunschweig 2013: Induction of growth of cells containing BBa_K1073035 on 2xYT agar plates containing ampicillin (0.29 mM) by synthetic autoinducer N-buturyl-HSL of different concentrations.
From left to right:Negative control (2xYT medium), 1 µM N-buturyl-HSL, 10 µM N-buturyl-HSL, and 100 µM N-buturyl-HSL.
Production of las autoinducer N-3-dodecanoyl-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 eforRed expression cassette is included in BBa_K1073035 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 1096
Illegal NheI site found at 1119
Illegal NheI site found at 2784
Illegal NheI site found at 2807 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2626
Illegal BamHI site found at 1391 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2188
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1866
| None |