Difference between revisions of "Part:BBa K1139150:Experience"
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<partinfo>BBa_K1139150 short</partinfo> | <partinfo>BBa_K1139150 short</partinfo> | ||
| − | + | ===1. Materials and Methods=== | |
| − | + | '''1-1. Construction'''<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> | ||
| − | -pSB6A1-Prm/lac-GFP (N99)…sample with CI | + | -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 expresses CI from its genome constitutively.<br> | ||
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[[Image:Titech2013_parts_K1139150_Fig1D.jpg|thumb|center|500px|]] | [[Image:Titech2013_parts_K1139150_Fig1D.jpg|thumb|center|500px|]] | ||
| − | + | '''1-2. Assay protocol'''<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 microL of the overnight cultures into LB (3 mL) containing antibiotics (Amp 50 µg/mL) and 1 mM of IPTG | + | 2. Take 30 microL 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 E. coli genome.<br> | We added IPTG in order to make sure to repress the expression of LacI derived from E. coli 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> | ||
| − | + | ===2. Results=== | |
Fig. 2-1 shows the fluorescence intensity detected by flow cytometer.<br> | Fig. 2-1 shows the fluorescence intensity detected by flow cytometer.<br> | ||
Fig. 2-2 is the extracted data which shows the comparison of N99 (IPTG+, with constitutive CI) and JM2.300 (IPTG+, without CI).<br> | Fig. 2-2 is the extracted data which shows the comparison of N99 (IPTG+, with constitutive CI) and JM2.300 (IPTG+, without CI).<br> | ||
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[[Image:Titech2013_parts_K1139150_Fig1C.jpg|thumb|center|500px|'''Fig. 2-2.''' Comparison of N99 and JM2.300]] | [[Image:Titech2013_parts_K1139150_Fig1C.jpg|thumb|center|500px|'''Fig. 2-2.''' Comparison of N99 and JM2.300]] | ||
| − | + | ===3. 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.<br> | 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.<br> | ||
We are now confirming that our ''rm/lac'' hybrid promoter is not only activated by CI but also repressed by LacI. | We are now confirming that our ''rm/lac'' hybrid promoter is not only activated by CI but also repressed by LacI. | ||
Revision as of 06:37, 26 September 2013
Prm/lac-GFP-TT
1. Materials and Methods
1-1. 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.
1-2. Assay protocol
1. Prepare overnight culture of each cell at 37°C for 12 hours.
2. Take 30 microL 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.
2. Results
Fig. 2-1 shows the fluorescence intensity detected by flow cytometer.
Fig. 2-2 is the extracted data which shows the comparison of N99 (IPTG+, with constitutive CI) and JM2.300 (IPTG+, without CI).
3. 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.
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