Part:BBa_K2267031
Plux4
Promoter activated by LuxR in concert with HSL
This part involves the -10 binding site, the -35 binding site, and the twenty nucleotides between that constitute the lux box. With this part, LuxR functions as a acyl-homoserine lactone-dependent repressor. LuxR responsed to the HSL produced by LuxI, N-(3-oxohexanoyl)-HSL. The Lux box is positioned such that it partially overlaps the consensus -35 and -10 hexamers of an RNA polymerase binding site. A quorum-sensing system involving LuxR, the transcriptional activator, and an acyl-homserine lactone signal regulate the lux operon in vibrio fischeri. In vibrio fischeri, the lux box, which is a 20-base inverted repeat unit, is positioned 42.5 bases upstream of the transcriptional start of the lux operon and is required for transcriptional activation.
Background information
Quorum-sensing bacteria such as Vibrio fischeri, are able to detected their own population density and implement density-based decision-making，Using the luxI/luxR quorum-sensing system, synthetic biologists have designed a large number of devices in prokaryotic microorganisms.Inevitably, high performance is required from such devices, and this includes reliability, sensitivity. Hence, to improve the properties of population-density switches, we design of ultrasensitive responses base https://parts.igem.org/Part:BBa_R0062 use Point mutation
Experiment Design
We selected the successful strains of the experiment and sequenced part1: https://parts.igem.org/Part:BBa_K2267029 part3: https://parts.igem.org/Part:BBa_K2267030 part4: https://parts.igem.org/Part:BBa_K2267031 part6: https://parts.igem.org/Part:BBa_K2267032 part8: https://parts.igem.org/Part:BBa_K2267033
Methods
The experiments for the characterization of parts were performed as described previously. For density-response testing, cells (E. coli strain BW25113) from single colonies on LB agar (BD, USA) plates were grown overnight in 1 ml nutrition-rich, acid-base equilibrium medium (REM) (15.2 g/l yeast extract (BD, USA), 0.5% (NH4)2SO4, 4 mM MgSO4, 2% glucose and 24 g/l K2HPO4.3H2O, and 9.6 g/l KH2PO4) in Falcon tubes overnight (8−12 h, 1000 rpm, 37 °C, mB100-40 Thermo Shaker, AOSHENG, China). The cultures were subsequently diluted 500-fold with REM in 96-well plates, which were further incubated at 37°C in a shaker at 1000 rpm. Once the diluted cultures reached an OD600 of 0.12–0.14 (~3 h), 10 μL aliquots were transferred into 1 mL REM in 24-well plates (Corning/Costar 3524). These plates were incubated at 37°C in a Varioskan Flash (Thermo Scientific, USA) under constant shaking at 1,000 rpm for 20 h to maintain exponential growth, during which the OD600 and fluorescence values were recorded
Results
We characterised the activation range of this device using a GFP reporter https://parts.igem.org/Part:BBa_K319039 . The results of our characterisation experiments can be found here
we did point mutation on https://parts.igem.org/Part:BBa_R0062 and the exact spots are
plux1: https://parts.igem.org/Part:BBa_K2267029
plux3: https://parts.igem.org/Part:BBa_K2267030
plux4: https://parts.igem.org/Part:BBa_K2267031
plux6: https://parts.igem.org/Part:BBa_K2267032
plux8: https://parts.igem.org/Part:BBa_K2267046
listed below. We linked gfp reporter genes luxr-plux1-gfp: https://parts.igem.org/Part:BBa_K2267043
luxr-plux3-gfp: https://parts.igem.org/Part:BBa_K2267041
luxr-plux4-gfp: https://parts.igem.org/Part:BBa_K2267042
luxr-plux6-gfp: https://parts.igem.org/Part:BBa_K2267044
luxr-plux8-gfp: https://parts.igem.org/Part:BBa_K2267045
on them respectively to test the function of modified promoters.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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