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 strain expresses CI from its genome constitutively.

2. Assay protocol
1. Prepare overnight cultures 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, measure the fluorescence intensity with a flow cytometer.

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 that of JM2.300 cells (CI-). From this result, we assume that our RM/lac hybrid promoter was actually activated by CI. In addition, N99 (IPTG-) showed lower fluorescence than that of N99 (IPTG+). From this result, we can assume that our RM/lac hybrid promoter was repressed by LacI derived from the E. coli genome.

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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