Team:NYMU-Taipei/Project/Promoter Strength Testing


 * All experiments were done in E. coli DH5alpha.
 * Maturation rate of GFP is 6.5mins [1]
 * All our analysis was done in excel documents: [[Image:NYMU-promotertesting.zip]] and done based on the paper by Leveau and Lindow [2].

Reporting Assay Protocol

 * 1) Single colonies for each biological replicate was selected from freshly streaked plates and cultured overnight in an 10ml tube of LB at 37C, 200rpm.
 * 2) Measure the OD600 and dilute it to 0.0325 in 3-5ml of LB (culture amount depends upon number of measurements. We usually used 3ml).
 * 3) Incubate it for an hour at 37C, 200rpm.
 * 4) Then for every hour:
 * 5) * Remove 250uL from the tube, put in a cuvette and measure the OD600.
 * 6) * Take 200uL from the cuvette and transfer it into a well on a black 96-well plate.
 * 7) * Measure the fluorescence in an ELIZA reader.

Conclusion

 * We have tested the relative strengths of p22, pCI, pLux (basal level), pLas (basal level), pTet, pPenI, and pLac.
 * We obtained relatively consistent results between the relative strengths of p22, pCI, pLux, and pLas, while obtaining not so consistent results between pTet, pPenI, and pLac.
 * We think it is due to the lack of experience earlier on and confusing us which relative strengths between pTet, pPenI, and pLac were correct.

Reference
[1] Megerle JA, Fritz G, Gerland U, Jung K, Radler JO: Timing and Dynamics of Single Cell Gene   Expression in the Arabinose Utilization System, Biophys J, 2008, 95(4): 2103–2115 [2] Leveau JHJ and Lindow SE, Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria, Journal of Bacteriology, 2001, 183(23): 6752-6762