Difference between revisions of "Part:BBa K1139150:Experience"
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-pSB6A1-Prm/lac-GFP (N99)…sample with CI<br> | -pSB6A1-Prm/lac-GFP (N99)…sample with CI<br> | ||
-pSB6A1--Prm/lac-GFP (JM2.300)…sample without CI<br> | -pSB6A1--Prm/lac-GFP (JM2.300)…sample without CI<br> | ||
| − | This N99 expresses CI from its genome constitutively.<br> | + | This N99 strain expresses CI from its genome constitutively.<br> |
[[Image:titech2013_parts_K1139150_exp_Fig1.jpg|frameless|none|250px||]] | [[Image:titech2013_parts_K1139150_exp_Fig1.jpg|frameless|none|250px||]] | ||
<b>2. Assay protocol</b><br> | <b>2. Assay protocol</b><br> | ||
| − | 1. Prepare overnight | + | 1. Prepare overnight cultures 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 | + | 3. After 4 hours of induction, measure the fluorescence intensity with a flow cytometer.<br> |
===Results=== | ===Results=== | ||
Revision as of 21:42, 28 October 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 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).
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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