Difference between revisions of "Part:BBa K1139150:Experience"
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<b>2. Assay protocol</b><br> | <b>2. Assay protocol</b><br> | ||
1. Prepare overnight culture of each cell at 37°C for 12 hours.<br> | 1. Prepare overnight culture of each cell at 37°C for 12 hours.<br> | ||
| − | 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)<br> | + | 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)<br> |
| − | We added IPTG in order to make sure to repress the expression of LacI derived from <i>E. coli</i> genome.<br> | + | *We added IPTG in order to make sure to repress the expression of LacI derived from <i>E. coli</i> genome.<br> |
3. After 4 hours of induction, flow cytometer measurements for GFP expression.<br> | 3. After 4 hours of induction, flow cytometer measurements for GFP expression.<br> | ||
Revision as of 02:24, 28 September 2013
Prm/lac-GFP-TT
Contents
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).
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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