"
Team:Tokyo Tech/cph
From 2009.igem.org
(→Method) |
(→The mechanism of photo-sensor) |
||
| Line 6: | Line 6: | ||
==The mechanism of photo-sensor== | ==The mechanism of photo-sensor== | ||
Figure 1 shows the mechanism of photo-sensor. Cph8 consists of cph1 and envZ. Cph1 is an extracellular sensor and cph1 bound to PCB (converted from heam by ho1 and pcyA) responds to the light. EnvZ is an intracellular response regulator and has a histidine kinase domain that phosphorylates ompR protein so that it can regulate gene expression. When cph8 is not exposed to the light, the histidine kinase domain phosphorylates ompR, and then ompR-phosphate activates the PompC by binding to it. When cph8 is exposed to the light, the histidine kinase can’t phosphorylate ompR so PompC is inactive. | Figure 1 shows the mechanism of photo-sensor. Cph8 consists of cph1 and envZ. Cph1 is an extracellular sensor and cph1 bound to PCB (converted from heam by ho1 and pcyA) responds to the light. EnvZ is an intracellular response regulator and has a histidine kinase domain that phosphorylates ompR protein so that it can regulate gene expression. When cph8 is not exposed to the light, the histidine kinase domain phosphorylates ompR, and then ompR-phosphate activates the PompC by binding to it. When cph8 is exposed to the light, the histidine kinase can’t phosphorylate ompR so PompC is inactive. | ||
| + | |||
| + | ==Experimental overview== | ||
| + | We attempted to construct Biobrick coding cph8 and PCB-related enzymes, assay them, and characterize them. We used the strain, Ru1012 [MC4100 ara+ \phi(OmpC-lacZ) 10-25 envZ::KanR], which chromosomally codes a ompC promoter fused to a lacZ reporter. That strain enables us to assay the activity of cph8 and PCB-related enzymes easily. Origin and resistance on the backbone is the most significant matter we have to consider, because we planned to transform Ru1012, putting two plasmids into it and the origins and resistances of the two plasmids should be different. Because Ru1012 codes KanR, we have to prepare two kinds of backbones. One codes AmpR, and the other codes Cm respectively. | ||
| + | |||
| + | ===The protocol for the lacZ assay=== | ||
| + | To quantitatively determine the performances of photo-receipting parts, we measured the amount of β-galactsidase expressed from lacZ reporter. | ||
| + | |||
| + | 1.Diluted overnight cultures of photo-receipting strains grown at 37℃ into 1:1000 2mL LB medium containing appropriate antibiotics in two 24-well plates | ||
| + | 2.Incubated two plates at 37℃ for 5 hours, one was exposed with 660nm LED lamp glowing at 7W, and the other was wrapped in aluminum foil and kept in darkness | ||
| + | 3.Diluted each strain into 1:100 in new wells | ||
| + | 4.Incubated two plates at 37℃ for another 5 hours, one was exposed with LED lamp, and the other was wrapped in aluminum foil and kept in darkness | ||
| + | 5.Spun 2mL of the final culture at 6000 rpm for 10 minutes in a microcentrifuge and then resuspended in 2mL of Z buffer | ||
| + | 6.Recorded the cell density by measuring the OD600 using spectrophotometer | ||
| + | 7.Diluted 0.5mL of the resuspended cells into 1mL in Z buffer and permeabilized by the addition of 100µL of chloroform and 50µL of 0.1% SDS | ||
| + | 8.vortexed and allowed to equilibrate at 28℃ for 5 minutes | ||
| + | 9.0.2mL of a solution containing 4.g/mL ONPG was added to the permeabilized cells | ||
| + | 10.Recorded the time when sufficient yellow color developed to be seen by eye, and stop the reaction by the adding 0.5mL of 1M Na2CO3 with vortexing | ||
| + | 11.Removed the chloroform by centrifuging 1mL of the quenched reaction at 13200 rpm for 5 minutes | ||
| + | 12.Measured OD420 and OD550 | ||
| + | 13.Calculated using the formula: | ||
| + | Z butter was prepared as follows: | ||
| + | H2O 500[mL] | ||
| + | Na2HPO4・12H2O 10.75[g] | ||
| + | NaH2PO4・2H2O 3.06[g] | ||
| + | KCl 0.375[g] | ||
| + | MgSO4・7H2O 0.123[g] | ||
| + | DTT 1[mL] | ||
==Result== | ==Result== | ||
==Discussion== | ==Discussion== | ||
Revision as of 05:06, 20 October 2009
| Main | Team | Terraforming | Experiments | [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Tokyo_Tech Parts] | Safety |
Contents |
Introduction
Mars also has day and night like Earth. The strength of the sunlight reaching Mars varies from day to night. During the day, the Mars’ surface temperature is about at most -20 degrees Celsius and in the night it drops to -100 degrees Celsius, so it natural that E. coli should behave differently in the day and night. Photo-sensor can be one powerful tool to control or regulate gene expression that is suitable for the temperature. For example, we introduced anti-freeze protein into E. coli, and it should express an appropriate amount of the protein based on the surface temperature, which is in proportion to the strength of light. We believe that this sensor can also be applied and utilized for terra-forming in some other way.
The mechanism of photo-sensor
Figure 1 shows the mechanism of photo-sensor. Cph8 consists of cph1 and envZ. Cph1 is an extracellular sensor and cph1 bound to PCB (converted from heam by ho1 and pcyA) responds to the light. EnvZ is an intracellular response regulator and has a histidine kinase domain that phosphorylates ompR protein so that it can regulate gene expression. When cph8 is not exposed to the light, the histidine kinase domain phosphorylates ompR, and then ompR-phosphate activates the PompC by binding to it. When cph8 is exposed to the light, the histidine kinase can’t phosphorylate ompR so PompC is inactive.
Experimental overview
We attempted to construct Biobrick coding cph8 and PCB-related enzymes, assay them, and characterize them. We used the strain, Ru1012 [MC4100 ara+ \phi(OmpC-lacZ) 10-25 envZ::KanR], which chromosomally codes a ompC promoter fused to a lacZ reporter. That strain enables us to assay the activity of cph8 and PCB-related enzymes easily. Origin and resistance on the backbone is the most significant matter we have to consider, because we planned to transform Ru1012, putting two plasmids into it and the origins and resistances of the two plasmids should be different. Because Ru1012 codes KanR, we have to prepare two kinds of backbones. One codes AmpR, and the other codes Cm respectively.
The protocol for the lacZ assay
To quantitatively determine the performances of photo-receipting parts, we measured the amount of β-galactsidase expressed from lacZ reporter.
1.Diluted overnight cultures of photo-receipting strains grown at 37℃ into 1:1000 2mL LB medium containing appropriate antibiotics in two 24-well plates 2.Incubated two plates at 37℃ for 5 hours, one was exposed with 660nm LED lamp glowing at 7W, and the other was wrapped in aluminum foil and kept in darkness 3.Diluted each strain into 1:100 in new wells 4.Incubated two plates at 37℃ for another 5 hours, one was exposed with LED lamp, and the other was wrapped in aluminum foil and kept in darkness 5.Spun 2mL of the final culture at 6000 rpm for 10 minutes in a microcentrifuge and then resuspended in 2mL of Z buffer 6.Recorded the cell density by measuring the OD600 using spectrophotometer 7.Diluted 0.5mL of the resuspended cells into 1mL in Z buffer and permeabilized by the addition of 100µL of chloroform and 50µL of 0.1% SDS 8.vortexed and allowed to equilibrate at 28℃ for 5 minutes 9.0.2mL of a solution containing 4.g/mL ONPG was added to the permeabilized cells 10.Recorded the time when sufficient yellow color developed to be seen by eye, and stop the reaction by the adding 0.5mL of 1M Na2CO3 with vortexing 11.Removed the chloroform by centrifuging 1mL of the quenched reaction at 13200 rpm for 5 minutes 12.Measured OD420 and OD550 13.Calculated using the formula: Z butter was prepared as follows: H2O 500[mL] Na2HPO4・12H2O 10.75[g] NaH2PO4・2H2O 3.06[g] KCl 0.375[g] MgSO4・7H2O 0.123[g] DTT 1[mL]
