Regulatory
pLux

Part:BBa_K2267029

Designed by: DENG, TINGYUE   Group: iGEM17_TUST_China   (2017-10-25)


Plux1

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 resonds 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 detect their own population density and regulate their growth accordingly.Using the luxI/luxR quorum-sensing system, synthetic biologists have designed a large number of devices in prokaryotic microorganisms.Naturally, high performance is required from such devices, and this includes reliability and sensitivity. Hence, with the goal to improve the properties of population-density switches, we design an ultrasensitive responses base. http://parts.igem.org/Part:BBa_R0062 use Point mutation

Experiment Design

T--TUST_China--part_Experiment_Design.png


part4: http://parts.igem.org/Part:BBa_K2267031

part3: http://parts.igem.org/Part:BBa_K2267030

part1: http://parts.igem.org/Part:BBa_K2267029

III 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 culture media 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 culture media reach a point where 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 at interval.

IV Results

We characterised the activation range of this device using a GFP reporter http://parts.igem.org/Part:BBa_K319039. The results of our characterisation experiments can be found here we did point mutation on http://parts.igem.org/Part:BBa_R0062 and the exact spots are

plux1: http://parts.igem.org/Part:BBa_K2267029

plux3: http://parts.igem.org/Part:BBa_K2267030

plux4: http://parts.igem.org/Part:BBa_K2267031

plux6: http://parts.igem.org/Part:BBa_K2267032

plux8: http://parts.igem.org/Part:BBa_K2267046

listed below. We linked gfp reporter genes luxr-plux1-gfp: http://parts.igem.org/Part:BBa_K2267043

luxr-plux3-gfp: http://parts.igem.org/Part:BBa_K2267041

luxr-plux4-gfp: http://parts.igem.org/Part:BBa_K2267042

luxr-plux6-gfp: http://parts.igem.org/Part:BBa_K2267044

luxr-plux8-gfp: http://parts.igem.org/Part:BBa_K2267045

on them respectively to test the function of modified promoters.

T--TUST_China--part_part_results.png Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None