This summer, our team is aiming to engineer bacteria for supplement and absorption of autoinducer-2 in the natural environment. We mainly design two cell machines: AI-2 Supplier is the cell machine which can directly supply and enrich the AI-2 molecular level; AI-2 Consumer is another cell machine which can sense, absorb and degrade the AI-2 in the environment. By taking advantage of the special characteristics of AI-2 controllers, we hope to directly control the population behaviors of bacteria in group levels.
What's more, biosafety is further taken into account in our project. By applying gene circuits to control essential gene expression under the assigned biocontainment conditions, we can blocks essential gene expression to kill the cell upon loss of the biocontainment signal.
To enrich the AI-2 concentration in the nature or artificial environment, we constructed two AI-2 Supplier Devices by overexpression of the components responsible for AI-2 production (luxS, mtn).
To 'quench' AI-2 signal in the nature or artificial environment, we constructed six AI-2 Consumer Devices by overexpression the components responsible for AI-2 uptake(lsrACDB), phosphorylation(lsrK) and degradation (lsrFG).
AI-2 Response Device
We successfully constructed two devices that could respond to AI-2 by producing GFP, to provide an independent means to use AI-2 Controller to alter heterologous gene expression. we show that a 1:1 mixture of AI-2 Response Device with AI-2 suppliers activated QS-activated GFP expression from the control group. And 1:1 mixture of AI-2 Response Device with AI-2 consumers could significantly depress QS-activated gene expression from the control cells.
This summer, we plan to mf-lon ssrA tag into 5 essential genes by using CRIPSR/Cas9 technology. By applying gene circuits to control mf-lon protease expression under the assigned biocontainment conditions, we can blocks essential gene expression to kill the cell upon loss of the biocontainment signal.
Demonstration of Experiment Results
We demonstrate utility of AI-2 Controllers by modulating naturally occurring processes of biofilm formation. We envision that ‘controller cells’ that modulate bacterial behavior by manipulating molecular communication, will find use in a variety of applications, particularly those employing natural or synthetic bacterial consortia.