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

1) Characterization of pLsrA -YFP:
The successfully ligated plasmid with pLsrA-YFP gene was first transformed into chemically competent LuxS(-) cells. The next day, one colony of cell with pLsrA-YFP plasmid was inoculated in 5ml LBA for 16 hours at 37oC and shaked at 225 rpm. Overnight cell culture was then centrifuged at 4000 rpm and 4oC for 10 minutes. The supernatant was discarded and cell pellets were re-suspended in 5ml of Ampicilin-containing M9 medium. The amount of M9 medium was adjusted until cell suspension had an OD600 of 1. The cell suspensions were then pipetted into 96-well microplate wells. This was followed by the adding of 50µl AI-2-containing supernatant into corresponding wells. There were 6 different supernatant solutions used, which correspond to the time points when they were obtained: 2, 3, 4, 5, 6, 8 hours. There were 3 different negative control samples being used for this study. First control sample contained cell suspension only. Second control sample was cell suspension with 50µl pure water added. And as the supernatants also contain large amount of LB, cell suspension with 50µl LB added was also used as another control sample. YFP measurement was carried out by VICTOR 3 multilabel reader at excitation wavelength of 490 nm and emission wavelength of 535 nm. Data were automatically gathered every 10 minutes and temperature was set at 37oC.

2) OD600 measurement of pLsrA-Lysis-containing LuxS mutant upon addition of AI-2:
One colony of LuxS(-) W3110 strain containing pLsrA-lysis plasmid was inoculated overnight in 50ml LB inside 250ml flask at 37 degrees C and 225rpm. After 16 hour inoculation, 0.5 ml of cell sample was diluted in 50ml fresh LB. Diluted cell sample was distributed into the wells of the black-96-well microplate with an amount of 200 µL per well. 50µl AI-2-containing supernatant was added into corresponding wells initially (set 1) and 1 hour after incubated inside absorbance-meter (set 2). Five different supernatant solutions used, which correspond to the time points when they had been obtained during AI-2 preparation: 3, 4, 5, 6, 8 hours.

Control samples included cell suspension alone; cell suspension with 50µl LB added initially (set 1) and after 1 hour incubation (set 2); cell suspension with 50µl water added initially (set 1) and after 1 hour incubation (set 2).