Part:BBa_K4294725
LuxI Transcriptional unit with BCD14 RBS (TetR Regulated)
The use of the TetR repressor system for the induction of the production of the luxI gene controlled by BCD14 RBS.
Figure 1: Comparison of 11 RBS, 240 min post-induction-DHSa-z1-cells
Usage and Biology
This induction system is borrowed from the antibiotic resistance mechanism encountered in some bacteria species. Due to its structure aTc, can activate this system without having antibiotic action. [1]
Circuit Design
TetR forms a dimer and binds to tet operators in pTet promoter and blocks the assembly of the transcription machinery. pTet is a medium-strength promoter that is constitutive “ON” in the absence of TetR. It contains two tet operators, one inside the core promoter sequence and the other after the -10 hexamer, leading to efficient repression. The system is efficiently induced via anhydrotetracycline hydrochloride (aTc), a tetracycline derivative which binds Tet R with an ~35-fold higher binding constant, being an effective inducer at very low concentrations. Additionally, its antibiotic activity is ~100-fold lower and has minimal toxic effect on E.coli cells in the concentration used for induction (photo design)The pTU1high copy plasmid was used for this construct.
Figure 2: Senders’ Circuit Overview. TetR is expressed by a constitutive promoter (BBa_J23106 and PlacI promoters were tested, please refer to Engineering Success for more regarding these trials) and the BBa_B0034 RBS. It binds to specific operators in the pTet promoter and blocks RNA polymerase binding and, thus, transcription.
Figure 3: The circuit at its “OFF” state (“0”)
Figure 4: The circuit at its “ON” state (“1”)
Measurment
For the measurement of the expression of the luxI gene, we incorporated in our design a 6*His tag that can be used either or immunological techniques (western blotting, ELISA) for the detection with anti-His antibody or for purification with affinity chromatography. Due to the luck of these resources our team performed an indirect assessment of this construct by inducing another population with the supernatant of E.coli (senders) that had this composite part and were induced with aTc for the production of the Acyl-Homoserine-Lactone Synthase. Last but not least we designed and assessed the luxI-36nt sfGFP constructs that are designed to estimate the production of luxI by the measurement of fluorescence. For more information visit these parts (BBa_K4294775).
Figure 5: DH5a-z1 luxI BCD14 only - BL21 Open loop ColE1
References
[1] Bertram R, Hillen W. The application of Tet repressor in prokaryotic gene regulation and expression. Microb Biotechnol. 2008 Jan;1(1):2-16. doi: 10.1111/j.1751-7915.2007.00001.x. PMID: 21261817; PMCID: PMC3864427.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 691
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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