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| − | '''1. Materials and Methods'''<br>
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| − | *1-1. Construction<br>
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| − | -pSB6A1-Ptet-GFP (N99)…positive control<br>
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| − | -pSB6A1-promoterless-GFP (N99)…negative control<br>
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| − | -pSB6A1-Prm/lac-GFP (N99)…sample with CI*<br>
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| − | -pSB6A1--Prm/lac-GFP (JM2.300)…sample without CI<br>
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| − | This N99 expresses CI from its genome constitutively.<br>
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| − | [[Image:Titech2013_parts_K1139150_Fig1D.jpg|thumb|center|500px|]]
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| − | *1-2. Assay protocol<br>
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| − | 1. Prepare overnight culture of each cell at 37°C for 12 hours.<br>
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| − | 2. Take 30 microL of the overnight cultures into LB (3 mL) containing antibiotics (Amp 50 µg/mL) and 1 mM of IPTG* (→fresh culture)<br>
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| − | We added IPTG in order to make sure to repress the expression of LacI derived from E. coli genome.<br>
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| − | 3. After 4 hours of induction, flow cytometer measurements for GFP expression.<br>
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| − | '''2. Results'''<br>
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| − | Fig. 2-1 shows the fluorescence intensity detected by flow cytometer.<br>
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| − | Fig. 2-2 is the extracted data which shows the comparison of N99 (IPTG+, with constitutive CI) and JM2.300 (IPTG+, without CI).<br>
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| − | [[Image:Titech2013_parts_K1139150_Fig1B.jpg|thumb|center|500px|'''Fig. 2-1.''' Fluorescence intensity detected by flow cytometer]]
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| − | [[Image:Titech2013_parts_K1139150_Fig1C.jpg|thumb|center|500px|'''Fig. 2-2.''' Comparison of N99 and JM2.300]]
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| − | '''3. Discussion'''<br>
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| − | N99 cells (CI+) showed higher fluorescence intensity than JM2.300 cells (CI-). From this experiment, we assume that our ''rm/lac'' hybrid promoter is actually activated by CI.<br>
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| − | We are now confirming that our ''rm/lac'' hybrid promoter is not only activated by CI but also repressed by LacI.
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