Difference between revisions of "Part:BBa K1139150:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | ||
how you used this part and how it worked out. | how you used this part and how it worked out. | ||
| + | |||
| + | '''1. Materials and Methods'''<br> | ||
| + | *1-1. Construction<br> | ||
| + | -pSB6A1-Ptet-GFP (N99)…positive control<br> | ||
| + | -pSB6A1-promoterless-GFP (N99)…negative control<br> | ||
| + | -pSB6A1-Prm/lac-GFP (N99)…sample with CI*<br> | ||
| + | -pSB6A1--Prm/lac-GFP (JM2.300)…sample without CI<br> | ||
| + | This N99 expresses CI from its genome constitutively.<br> | ||
| + | |||
| + | [[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> | ||
| + | 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> | ||
| + | 3. After 4 hours of induction, flow cytometer measurements for GFP expression.<br> | ||
| + | |||
| + | '''2. Results'''<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> | ||
| + | |||
| + | [[Image:Titech2013_parts_K1139150_Fig1B.jpg|thumb|center|500px|'''Fig. 2-1.''' Fluorescence intensity detected by flow cytometer]] | ||
| + | [[Image:Titech2013_parts_K1139150_Fig1C.jpg|thumb|center|500px|'''Fig. 2-2.''' Comparison of N99 and JM2.300]] | ||
| + | |||
| + | '''3. Discussion'''<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. | ||
| + | |||
===Applications of BBa_K1139150=== | ===Applications of BBa_K1139150=== | ||
Revision as of 03:12, 26 September 2013
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
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.
Applications of BBa_K1139150
User Reviews
UNIQ3340275b7026a8fe-partinfo-00000000-QINU UNIQ3340275b7026a8fe-partinfo-00000001-QINU