Team:Tokyo Tech/Consortium

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[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Tokyo_Tech Parts]

Safety

Achievement

We confirmed the temperature-regulated function of lambda promoter, lux signaling system.

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?

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 specialized 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 specialized microorganism(cyanobacteria,iron-oxidizing bacteria...etc).

Temperature regulated luxI

Simple model of microbial consortium

We constructed plasmid that contains temperature-regulated luxI as a key signaling　device.

This system uses a temperature sensitive variant of cI lambda repressor to regulate the lambda promoter. E.coli N4830 strain expresses temperature-sensitive cI protein constitutively.

Temperature sensing of lambda promoter

BBa_K193000(pL-GFP) BBa_I763007(Bologna07,pL-mRFP)

[fig]

We confirmed the temperature-regulated function of lambda promoter.

We introduced GFP or mRFP reporter regulated by cI to E.coli N4830 strain expressing temperature-sensitive cI protein. E.coli express GFP or mRFP at 37℃, but don’t at 27℃. This result suggests that the lambda promoter works and regulates GFP or mRFP expression.

Temperature sensing of 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 produce AHL under conditions of high temperature, E.coli express GFP.

We can expect E.coli express GFP at 37℃, but don’t at 27℃. This result suggests that E.coli express luxI at 37℃, but don’t at 27℃. And signaling device works and regulates by temperature.

Assay

To quantitatively determine the performances of sensor parts in the receiver plasmid, the change of fluorescence intensities of the receiver strains in the change of were measured.

Dilute overnight cultures of E.coli strains grown at 37℃ in LB medium containing appropriate antibiotics 1:100 in LB medium.(prepare 2 fresh cultures in each sample)
Incubate at 37℃ or 27℃ as fresh cultures for 6 hours＊
Take 1 ml of each culture to 2 ml tube.
Centrifuge for 1 min at 9000 rpm.
Discard the supernatant with a pipette.
Dissolve the pellet at the bottom of the tube in PBS to dilute samples to OD=0.8.

Take 200 μl of the washed culture to 96-well plate and measure its fluorescence intensity by Fluor meter (FLA).

＊experiment3: 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+)

experiment1　pL-GFP temperature-regulated assay

The measured fluorescence intensity was corrected by subtracting the background fluorescence, measured in control wells containing 200 μl 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-GFP, which expresses GFP constitutively, was used as a positive control. A strain, transformed with PlacIq -melA, which does not express GFP was used as a negative control.

experiment2　pL-mRFP temperature-regulated assay

The measured fluorescence intensity was corrected by subtracting the background fluorescence, measured in control wells containing 200 μl 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-mRFP, which expresses RFP constitutively, was used as a positive control. A strain, transformed with promoterlessRBS-GFP, 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 measured fluorescence intensity was corrected by subtracting the background fluorescence, measured in control wells containing 200 μl 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-GFP, which expresses GFP constitutively, was used as a positive control. A strain, transformed with PtetR-luxR-TT-PluxR-luxR-GFP(AHL+), which expresses GFP, was used as a positive control. A strain, transformed with PtetR-luxR-TT-PluxR-luxR-GFP(AHL-), which does not expresses GFP, was used as a negative control. A strain, transformed with PlacIq-melA, which does not express GFP was used as a negative control.

We confirmed that the part pL-mRFP (Bologna07), which did not use as a temperature–regulated system before, could use also for this system.

@@ Line 29: / Line 29: @@
 We constructed plasmid that contains temperature-regulated luxI as a key signaling　device.
-This system uses a temperature sensitive variant of cI lambda repressor to regulate the lambda promoter. E.coli N4830 strain expresses temperature-sensitive cI protein constitutively.
+This system uses a temperature sensitive variant of cI lambda repressor to regulate the lambda promoter. ''E.coli'' N4830 strain expresses temperature-sensitive cI protein constitutively.
 ===Temperature sensing of lambda promoter===
@@ Line 40: / Line 40: @@
 We confirmed the temperature-regulated function of lambda promoter.
-We introduced GFP or mRFP reporter regulated by cI to E.coli N4830 strain expressing temperature-sensitive cI protein. E.coli express GFP or mRFP at 37℃, but don’t at 27℃. This result suggests that the lambda promoter works and regulates GFP or mRFP expression.
+We introduced GFP or mRFP reporter regulated by cI to ''E.coli'' N4830 strain expressing temperature-sensitive cI protein. ''E.coli'' express GFP or mRFP at 37℃, but don’t at 27℃. This result suggests that the lambda promoter works and regulates GFP or mRFP expression.
 ===Temperature sensing of lux signaling===
@@ Line 49: / Line 49: @@
 [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 produce AHL under conditions of high temperature, E.coli express GFP.
+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.
-We can expect E.coli express GFP at 37℃, but don’t at 27℃.
+We can expect ''E.coli'' express GFP at 37℃, but don’t at 27℃.
 This result suggests that E.coli express luxI at 37℃, but don’t at 27℃. And signaling device works and regulates by temperature.