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.

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 terraforming of Mars?

In Mars, [http://partsregistry.org/Part:BBa_K193000 as described above], 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

Simple model of microbial consortium

We considered interaction between two species as a microbial consortium model.
We use Master E.coli and specialized bacteria like iron-oxidizing bacteria.
Standard bacteria have signaling devices regulated by sensors and logic gates. Meanwhile, specialized bacteria have additional function that is activated by signals.

Temperature sensing using lux signaling

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.

[http://partsregistry.org/Part:BBa_K193001 BBa_K193001] (CI repressible LuxI generator)
[http://partsregistry.org/Part:BBa_J54140 BBa_J54140] (LuxR generator)

We applied luxI signaling to temperature-regulated system. We introduced luxI signaling device and GFP reporter to E.coli N4830 strain. In this system, if E.coli produces AHL under conditions of high temperature, E.coli expresses GFP.

Materials and Methods

Reporter Assay

Each srain was grown overnight(~12 hours ) at 37℃ and shaken in 3ml LB. Then two sets of fresh culture were made by diluting aliquots of the overnight culture with LB medium to OD600 = 0.1. One set of the culture is incubated at 37℃ while the other set is incubated at 27℃ for 6 hrs. 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　BBa_K193000 (GFP reporter regulated by CI)

The target strain was N4830 transformed [http://partsregistry.org/Part:BBa_K193000 BBa_K193000] (GFP reporter regulated by CI).

Positive control strain was N4830 transformed with [http://partsregistry.org/Part:BBa_K121010 BBa_K121010] (Ptet-GFP), which expresses GFP constitutively.
Negative control strain was N4830 transformed with [http://partsregistry.org/Part:BBa_K193601 BBa_K193601] (PLacIq-RBS-melA), which does not express GFP.

experiment2　BBa_I763007(mRFP reporter regulated by CI)

The target strain was N4830 transformed with [http://partsregistry.org/Part:BBa_I763007 BBa_I763007] (mRFP reporter regulated by CI).

Positive control strain was N4830 transformed with [http://partsregistry.org/Part:BBa_I13521 BBa_I13521] (Ptet-mRFP), which expresses mRFP constitutively.
Negative control strain was N4830 transformed with [http://partsregistry.org/Part:BBa_I13504 BBa_I13504] (promoterless-gfp), which does not express mRFP.

experiment3　BBa_K193001 (CI repressible LuxI generator)

The target strain was N4830 transformed with [http://partsregistry.org/Part:BBa_K193001 BBa_K193001] (CI repressible LuxI generator) and [http://partsregistry.org/Part:BBa_J54140 BBa_J54140] (LuxR generator).

Positive control strain was N4830 transformed with [http://partsregistry.org/Part:BBa_J54140 BBa_J54140], which expresses GFP. (AHL+)
Negative control-1 strain was N4830 transformed with [http://partsregistry.org/Part:BBa_J54140 BBa_J54140], which leaky expresses GFP. (AHL-)
Negative control-2 strain was N4830 transformed with [http://partsregistry.org/Part:BBa_K193001 BBa_K193001], which does not express GFP.

Result

[fig:PL-GFP]

[fig:PL-mRFP]

[fig:PL-luxI+luxR]

luxR(AHL+),luxR(AHL-) ; strain N4830 transformed only with luxR expressing plasmid

luxR+luxI; strain N4830 transformed with luxR expressing plasmid and luxI expressing plasmid

luxI; strain N4830 transformed with luxI expressing plasmid

In the strain with luxR, GFP expression was observed dependent on the presence of AHL. In the strain with both of luxR and luxI, difference in GFP expression was observed (at 27℃ and at 37℃.) dependent on temperature. The culture(strain with luxI and luxR) grown at 37℃ showed about 2.5 fold higher fluorescence intensity than that grown at 27℃. This result indicates that the pL promoter and luxI presumably worked properly in the luxI expressing plasmid we made.

Discussion

http://partsregistry.org/Part:BBa_K193000

Reference

Katie Brenner, Lingchong You and HFrances H. Arnord, Engineering microbial consortia: a new frontier in synthetic biology, Trends in Biotechnology, 2008

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 ===Reporter Assay===
-We diluted overnight cultures of ''E.coli'' strains grown at 37℃ in LB medium containing appropriate antibiotics 1:100 in LB medium.
+Each srain was grown overnight(~12 hours ) at 37℃ and shaken in 3ml LB. Then two sets of fresh culture were made by diluting aliquots of the overnight culture with LB medium to OD600 = 0.1.  One set of the culture is incubated at 37℃ while the other set is incubated at 27℃ for 6 hrs.
-We prepared 2 fresh cultures in each sample.
-We incubated at 37℃ and 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.