A composite part of which GFP expression can directly respond to AI-2 concentration in the natural or artificial environment
BBa_K1981202
Autoinducer-2 Response Device B
A composite part which has a tighter regulation and delayed response compared to AI-2 Response Device A
1. Best New Basic Part: BBa_K1981101
lsr promoter of LuxS/AI-2 signaling pathway in E. coli (BBa_K1981101)
AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In a group of bacteria exemplified by E. coli MG1655, AI-2 response involves lsr gene clusters that encode lsrACDB, lsrK, lsrFG. plsr is the promoter of the lsr operon.
We isolated the promoter region of the lsr operon to regulate the expression of the report gene, GFP (BBa_E0040). In order to test the function of the lsr promoter, we transformed the plasmid pTrcHisB containing plsr with GFP gene at its downstream into E. coli MG1655 ΔluxS. We directly add exogenous AI-2 into the culture. The final concentraton of AI-2 is 50μM. Every one hour, optical density was measured and samples were harvested for HPLC analysis. As you can see, after induced by AI-2, the GFP expression is increased compared to control group.
Figure 1.1: GFP expression after promoter lsr is induced by exogenous AI-2
Then we test whether promoter lsr can respond to different AI-2 concentration. We directly add exogenous AI-2 into the culture. The final concentraton of AI-2 is 50μM, 40μM, 30μM, 20μM, 10μM, 0μM. Every one hour, optical density was measured and samples were harvested for HPLC analysis. The result below shows that promoter lsr can respond to different AI-2 concentration resulting in different GFP expression.
Figure 1.2: GFP expression when add exogenous AI-2
2. Best New Composite Part: BBa_K1981201
Autoinducer-2 Response Device A
This composite part consists of the AI-2 (autoinducer-2) quorum sensor-inducible promoter BBa_K1981101, a GFP coding sequence BBa_E0040, a double terminator BBa_B0015. We firstly isolated promoter lsr from E.coli MG1655. GFP BBa_E0040 and double terminator BBa_B0015 are standard part offered by iGEM. Then we successfully constrcuted plsr+GFP+double terminator using homologous recombination technology.
In AI-2 Response Device A, GFP expression is under the control of promoter, lsr. When phospho-AI-2 binds LsrR, expression of GFP ensues. The expression of GFP can directly respond to the AI-2 level in the environment, which is an alternative way to reflect the AI-2 concentration in the natural or artificial environment.
We fisrtly tested whether AI-2 Response Device A can respond to different AI-2 concentration. We directly added exogenous AI-2 into the culture. The final concentraton of AI-2 is 50μM, 40μM, 30μM, 20μM, 10μM, 0μM. Every one hour, optical density was measured and samples were harvested for HPLC analysis. The result below shows that deicve can respond to different AI-2 concentration resulting in different GFP expression.
Figure 2.1: GFP expression of AI-2 Response Device A when add exogenous AI-2.
AI-2 Consumers was constructed by iGEM 2016 NKU_China by overexpression the components responsible for AI-2 uptake (lsrACDB), phosphorylation (lsrK), and degradation (lsrFG), which can directly absorb and degrade AI-2 in the nature or artificial environment. When E.coli consisting of AI-2 Response Device A are co-cultured with AI-2 Consumers, the GFP expression of AI-2 Response Device A is directly decreased compared to control group.
Figure 1.2: GFP expression of AI-2 Response Device A when co-cultured with AI-2 Consumers.
AI-2 Suppliers was constructed by iGEM 2016 NKU_China by overexpression the components responsible for AI-2 production (luxS, mtn), which can directly supply and enrich the AI-2 molecular level in the nature or artificial environment. When E. coli consisting of AI-2 Response Device A are co-cultured with AI-2 Suppliers, the GFP expression of AI-2 Response Device A is directly increased compared to control group.
Figure 2.3: GFP expression of AI-2 Response Device A when co-cultured with AI-2 Suppliers.
