Difference between revisions of "Part:BBa K2267031"
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Denise Deng (Talk | contribs) (→Results) |
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===Background information=== | ===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 | + | 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=== | ===Experiment Design=== | ||
| Line 25: | Line 26: | ||
===Methods=== | ===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 | + | 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=== | ===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 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. | ||
| + | https://static.igem.org/mediawiki/2017/e/e6/T--TUST_China--part_part_results.png | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Latest revision as of 03:55, 2 November 2017
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]