Team:NTU-Singapore/Wetlab/Protocols

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NTU@iGEM Project Introduction Methodology Team Introduction Team background Notebook Dry Lab Introduction ODEs used Modeling Constructs Parameters used Deterministic Modeling Sensitivity Analysis Stochastic Modeling Wetlab Introduction Materials and Equipment Protocols Verification of Detection & Lysis System Our New Parts Characterization Introduction Standard Promoters Characterization of pLacI-GFP pLacI-GFP images and clips Characterization of pLsrA-YFP New characterization Proposal Parameter Estimation & Correlations Acknowledgements

I) MATERIALS:
1) Preparation of AI-2:
Pure AI-2 is very difficult to harvest or synthesize. However, as a quorum sensing messenger, AI-2 is present in intercellular medium at considerable amount. In this project, AI-2 was indirectly harvested by obtaining cell supernatants at different time points of cell growth. Glucose was added to the growth medium as it promotes AI-2 production significantly (Liang Wang, 2005).

A colony of E. coli W3110 was grown overnight in LB for 16 hours at 37 oC. The overnight culture was then diluted in LB plus 0.8% glucose to an optical density at 600 nm (OD600) of 0.02. The cells were incubated at 37oC with shaking at 225 rpm in 500ml Erlenmeyer flask. Cell aliquots were removed at each time point for measurement of the OD600. The measured values were shown in the following table: