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Team:Tokyo Tech/Consortium
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We discarded the supernatant with a pipette. We dissolved the pellet at the bottom of the tube in PBS to dilute samples to OD=0.8. | We discarded the supernatant with a pipette. We dissolved the pellet at the bottom of the tube in PBS to dilute samples to OD=0.8. | ||
We took 200 μl of the washed culture to 96-well plate and measure its fluorescence intensity by Fluorescent Image Analyzer(FLA) and Fluorescence Activated Cell Sorting(FACS). | We took 200 μl of the washed culture to 96-well plate and measure its fluorescence intensity by Fluorescent Image Analyzer(FLA) and Fluorescence Activated Cell Sorting(FACS). | ||
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| + | The measured fluorescence intensity was normalized by subtracting the background fluorescence intensity of PBS. The corrected value was normalized to culture volume and OD600 and expressed in fluorescence per (ml x OD600). | ||
===experiment1 pL-GFP temperature-regulated assay=== | ===experiment1 pL-GFP temperature-regulated assay=== | ||
Revision as of 12:10, 21 October 2009
| Main | Team | Terraforming | Experiments | [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Tokyo_Tech Parts] | Safety |
Contents |
Achievement
We confirmed the temperature-regulated function of lambda promoter, lux signaling system.
Introduction~Why do we need Microbial consortium?~
What’s Microbial consortium?
Microbial consortium is a group of different species of microorganisms that are interacting with each other. For instance, there are predator-prey interactions, communications and so on.
Why do we need Microbial consortium for terra-forming of Mars?
In Mars, as described above
Link
,there are much carbon dioxide and carbonate. However there is little amount of organic matter. Heterotrophic microorganism like E.coli cannot survive on its own. That's why autotrophic microorganisms are needed for the production of organic compounds in early stage of terra-forming.
In order to survive harsh conditions on Mars, bacteria should be adapted to environmental change. Therefore, Introducing various sensing elements to bacteria can be one solution, such as temperature sensor, light sensor, pH sensor and so on. It is difficult to introduce intricate genetic circuitry to autotrophic microorganism(cyanobacteria,iron-oxidizing bacteria...etc), on the other hand intricate genetic circuitry can be introduced to E.coli easily.
Our approach is the following.
Establishing consortium can give benefit both to E.coli and to autotrophic microorganism(cyanobacteria,iron-oxidizing bacteria...etc).
A simple model of microbial consortium
We constructed plasmid that contains temperature-regulated luxI as a key signaling device.
This system uses a temperature-sensitive cI repressor to regulate the lambda promoter. E.coli N4830 strain expresses temperature-sensitive cI protein constitutively.
Temperature sensing using lux signaling
BBa_K193001(pL-luxI) BBa_J54140(luxR,Tokyo_Alliance06)
[fig]
We applied luxI signaling to temperature-regulated system. We introduced luxI signaling device and GFP reporter to E.coli N4830 strain expressing temperature-sensitive cI protein. In this system, if E.coli produces AHL under conditions of high temperature, E.coli expresses GFP.
This figure shows E.coli express GFP at 37℃, but not to at 27℃. This result suggests that E.coli expresses luxI at 37℃, but don’t at 27℃. And signaling device works and regulates by temperature.
Materials and Methods
Protocol-Assay-
We diluted overnight cultures of E.coli strains grown at 37℃ in LB medium containing appropriate antibiotics 1:100 in LB medium. We prepared 2 fresh cultures in each sample. We Incubated at 37℃ or 27℃ as fresh cultures. After 6 hours, We took 1 ml of each culture to 2 ml tube, and centrifuged for 1 min at 9000 rpm. We discarded the supernatant with a pipette. We dissolved the pellet at the bottom of the tube in PBS to dilute samples to OD=0.8. We took 200 μl of the washed culture to 96-well plate and measure its fluorescence intensity by Fluorescent Image Analyzer(FLA) and Fluorescence Activated Cell Sorting(FACS).
The measured fluorescence intensity was normalized by subtracting the background fluorescence intensity of PBS. The corrected value was normalized to culture volume and OD600 and expressed in fluorescence per (ml x OD600).
experiment1 pL-GFP temperature-regulated assay
- The target strain was N4830 transformed BBa_K193000(pL-GFP) on pSB6A1.
[fig]
The measured fluorescence intensity was normalized by subtracting the background fluorescence intensity of PBS. The corrected value was normalized to culture volume and OD600 and expressed in fluorescence per (ml x OD600).
- A strain, transformed with BBa_K121010(PtetR-GFP) on pSB6A1, which expresses GFP constitutively, was used as a positive control.
- A strain, transformed with BBa_K193603(PlacIq -melA) on pSB6A1, which does not express GFP was used as a negative control.
experiment2 pL-mRFP temperature-regulated assay
- The target strain was N4830 transformed with BBa_I763007(pL-mRFP, Bologna07) on pSB1A2.
[fig]
The measured fluorescence intensity was normalized by subtracting the background fluorescence intensity of PBS. The corrected value was normalized to culture volume and OD600 and expressed in fluorescence per (ml x OD600).
- A strain, transformed with BBa_I13521(PtetR-mRFP) on pSB1A2, which expresses RFP constitutively, was used as a positive control.
- A strain, transformed with BBa_13504(promoterless-RBS-GFP) on pSB 1A2, which does not express mRFP was used as a negative control.
http://partsregistry.org/Part:BBa_I763007:Experience
experiment3 pL-luxI temperature-regulated assay
- The target strain was N4830 transformed with BBa_K193001(pL-luxI) on pSB6A1 and BBa_J54140(luxR,Tokyo_Alliance06) on pSB3K3
[fig]
The measured fluorescence intensity was normalized by subtracting the background fluorescence intensity of PBS. The corrected value was normalized to culture volume and OD600 and expressed in fluorescence per (ml x OD600).
- A strain, transformed with PtetR-luxR-TT-PluxR-luxR-GFP(AHL+), which expresses GFP, was used as a positive control.
This strain was N4830 transformed with pBR322(AmpR).
- A strain, transformed with PtetR-luxR-TT-PluxR-luxR-GFP(AHL-), which does not expresses GFP, was used as a negative control.
This strain was N4830 transformed with pBR322(AmpR).
- A strain, transformed with PlacIq-melA, which does not express GFP was used as a negative control.
This strain was N4830 transformed with (KanR).
＊After their OD600 reach 0.2, add 0.25 μM AHL 6 μl (only a strain, transformed with PtetR-luxR-TT-PluxR-luxR-GFP,AHL+)
Result
Discussion
- We confirmed the temperature-regulated function of lambda promoter.
We introduced GFP and mRFP reporter regulated by cI to E.coli N4830 strain (expressing temperature-sensitive cI protein). The figure shows E.coli express GFP and mRFP at 37℃, but not to at 27℃. This result suggests that the lambda promoter works and regulates GFP or mRFP expression.
- We confirmed that the part pL-mRFP (Bologna07), which did not use as a temperature–regulated system before, could use also for this system.
- We applied luxI signaling to temperature-regulated system. We introduced luxI signaling device and GFP reporter to E.coli N4830 strain expressing temperature-sensitive cI protein. In this system, if E.coli produce AHL under conditions of high temperature, E.coli express GFP.
Reference
- Katie Brenner, Lingchong You and HFrances H. Arnord, Engineering microbial consortia: a new frontier in synthetic biology, Cell, 2008
