Part:BBa_K1139150:Experience

Prm/lac-GFP-TT

Materials and Methods

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.

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.

Results

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

Fig. 1. Fluorescence intensity detected by flow cytometer

Fig. 2. Comparison of N99 and JM2.300

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.

For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/RM-lac_Hybrid_Promoter_Assay our work in Tokyo_Tech 2013 wiki].

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