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Revision as of 17:47, 21 October 2019
Demonstrate
Experiment
To test the effectiveness of our new part luxI promoter with FUR - we needed to expose cells containing transformed plasmid into both iron rich and iron starved environments. Single colonies were inoculated in 50 ml LB broth containing Ampicillin in a 1000:1 ratio and 40 μM FeSO4 in Falcon tubes and cultured at 37 C until OD600 = 0.5. 10ml culture was added to each of three 15ml tubes. Sample A contains blank cell (without plasmid) culture. Sample B contains culture (with plasmid) with 200 μM DP (2,2'-Dipyridine). The function of the 2,2'-Dipyridine is to remove iron in the cellular environment and thus mimic the low iron environment of the gut. Sample C contains only the culture (with plasmid) without any 2,2'-Dipyridine.
After induction with DP for 4 hours, 1 ml of each cell culture broth was transferred to two 1.5 ml sterile centrifuge tubes and centrifuged at 4000rpm for 4 minutes. After removing the supernatant, we wash the cell with PBS buffer. Then, 100 μM culture was added into 96 well white polystyrene microplate and black polystyrene microplate, each with three samples. We measured the OD600 and Fluorescence (Excitation: 485nm/ Emission: 528nm) by using plate reader. The data was recorded. After that, we calculate the average OD600 and Fluorescence for each sample. For each of samples, we divided the relative fluorescence value (RFV) by the average OD600. This quantitative test was used to determine Fur and luxI/luxR-controlled protein expression under iron deprivation in E. coli.
- Sample A = Blank (E.coliBL21(DE3) cells with no plasmid)
- Sample B = E.coliBL21(DE3) cells containing our ironQS system (BBa_K3031016) and grown in iron rich media PLUS iron chelator 2,2'-Dipyridine
- Sample C = E.coliBL21(DE3) cells containing our ironQS system (BBa_K3031016) and grown in iron rich media only.
Blank | Iron QS+DP | Iron QS | |
---|---|---|---|
RFV(AVG) | 952237 | 397057 | 554270 |
OD(AVG) | 0.604 | 0.119 | 0.343 |
RFV/OD600 | 1576551.325 | 3345985.955 | 1614379.612 |