Part:BBa_K1139150:Experience

Prm/lac-GFP-TT

1. Materials and Methods

1-1. Construction
-pSB6A1-Ptet-GFP (N99)…positive control
-pSB6A1-promoterless-GFP (N99)…negative control
-pSB6A1-Prm/lac-GFP (N99)…sample with CI
-pSB6A1--Prm/lac-GFP (JM2.300)…sample without CI
This N99 expresses CI from its genome constitutively.

1-2. Assay protocol
1. Prepare overnight culture of each cell at 37°C for 12 hours.
2. Take 30 µL of the overnight cultures into LB (3 mL) containing antibiotics (Amp 50 µg/mL) and 1 mM of IPTG (→fresh culture)
We added IPTG in order to make sure to repress the expression of LacI derived from E. coli genome.
3. After 4 hours of induction, flow cytometer measurements for GFP expression.

2. Results

Fig. 2-1 shows the fluorescence intensity detected by flow cytometer.
Fig. 2-2 is the extracted data which shows the comparison of N99 (IPTG+, with constitutive CI) and JM2.300 (IPTG+, without CI).

Fig. 2-1. Fluorescence intensity detected by flow cytometer

Fig. 2-2. Comparison of N99 and JM2.300

3. Discussion

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.
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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