Part:BBa_K3788019
Expression of GFP and of the timer device under the control of the LexA-controlled promoter
The Part BBa_K3788019 is a part composed with BBa_K3788017 part (lexA-repressed promoter), BBa_K3788018 (timer lysis devise), et de BBa_E0040 (GFP) fused to caa.
With this design, this part integrates a part of the caa gene, coding for the Colicin A toxin. It is also composed from cal and cai genes, respectively for the lysis protein CaL and the immunity protein Cal.
In normal conditions, caa and cai genes are positioned as an operon, and the cai gene is located on the antisense strand and expressed in a constitutive manner.
This system’s regulation is constituted from many regulation elements, which permits it to obtain a fine-tuned regulation.
This system is located on a plasmid present in some E. Coli populations. It allows Colicin A accumulation on the host cell, and then induces its death due to CaL action, which provokes Colicin A liberation on the extracellular medium, which induces the death of bacteria that don’t produce Cal protein.
cal gene codes for CaL protein, a lipopeptide with a length between 28 and 48 amino acid residues. This protein is produced as a precursor and then processed. Once the protein is processed, it can form pores in the bacterial membrane, which leads to the quasi-lysis of the host cell, and provokes a leak from the cellular component into the medium.
Figure : schematic representation of the construction with the different coding sequences, restriction sites used to insert the GFP coding sequence, and the known terminators.
Methods
In order to characterize the promoter strength, we fused it to the GFP encoding sequence BBa_E0040 and to the timer-lysis device part BBa_K3788018 (composite part BBa_K3788019). Induction of the SOS response was induced by mitomycin C treatments and the fluorescence was recorded in a dose-dependent manner using a microplate reader. Following iGEM Measurement recommendations, the GFP fluorescence intensity was converted to a concentration of fluorescein protein thanks to a standard curve of fluorescein dilution series.
Results
Figure:Measure of the GFP signal (as fluorescein equivalent) of bacterial liquid cultures expriming BBa_K3788019 across time with different quantities of MitomycinC added to the growing medium.
The experiment was done in triplicate. Standard deviation is small and not visible on the graph.
Our data confirmed that the promoter part BBa_K3788017 responds to mitomycin in a dose dependent manner from 50 to 800 ng.ml-1. The signal is significantly detected 90 min to 130 min after the beginning of the experiment, depending of the inducer tested concentration.
Moreover, we showed that the promoter is strongly repressed in the absence of the inducer, as no fluorescence signal is detected for the untreated control during the time course of the experiment (450 min).
Finally, thanks to the composite part BBa_K3788019, we demonstrated that during the 90 to 210 min window after induction, there is a linear relationship between the accumulation of GFP signal and Mitomycin concentration. We established titration curves for this time period (R2>0.975).
Figure:Linear relationship between mitomycin concentration and GFP signal 90 and 210 minutes after induction
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1528
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1848
Illegal BamHI site found at 1521
Illegal XhoI site found at 1397 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1597
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 867
Illegal SapI.rc site found at 1351
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