Difference between revisions of "Part:BBa K1139150:Experience"
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<partinfo>BBa_K1139150 short</partinfo> | <partinfo>BBa_K1139150 short</partinfo> | ||
| − | === | + | ===Materials and Methods=== |
| − | + | <b>1. Construction</b><br> | |
-pSB6A1-Ptet-GFP (N99)…positive control<br> | -pSB6A1-Ptet-GFP (N99)…positive control<br> | ||
-pSB6A1-promoterless-GFP (N99)…negative control<br> | -pSB6A1-promoterless-GFP (N99)…negative control<br> | ||
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This N99 expresses CI from its genome constitutively.<br> | This N99 expresses CI from its genome constitutively.<br> | ||
| − | [[Image:Titech2013_parts_K1139150_Fig1D.jpg|frameless|left|500px|]] | + | [[Image:Titech2013_parts_K1139150_Fig1D.jpg|frameless|left|500px|]] |
| − | + | <br><br><br><br><br><br><br><br><br><br><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 ( | + | 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 | + | 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> | ||
| − | === | + | ===Results=== |
| − | Fig. | + | Fig. 1 shows the fluorescence intensity detected by flow cytometer.<br> |
| − | Fig. | + | Fig. 2 is the extracted data which shows the comparison of N99 (IPTG+, with constitutive CI) and JM2.300 (IPTG+, without CI).<br> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | [[Image:Titech2013_parts_K1139150_Fig1B.jpg|thumb|center|500px|<b>Fig. 1.</b> Fluorescence intensity detected by flow cytometer]] | |
| − | + | [[Image:Titech2013_parts_K1139150_Fig1C.jpg|thumb|center|500px|<b>Fig. 2.</b> 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 <i>rm/lac</i> hybrid promoter is actually activated by CI.<br> | ||
| + | We are now confirming that our <i>rm/lac</i> hybrid promoter is not only activated by CI but also repressed by LacI. | ||
===Applications of BBa_K1139150=== | ===Applications of BBa_K1139150=== | ||
Revision as of 17:38, 26 September 2013
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).
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.
Applications of BBa_K1139150
User Reviews
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