Difference between revisions of "Part:BBa K1139150:Experience"
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__NOTOC__
 
 
<partinfo>BBa_K1139150 short</partinfo>
 
<partinfo>BBa_K1139150 short</partinfo>
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__TOC__
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===1. Materials and Methods===
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===Materials and Methods===
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'''1-1. Construction'''<br>
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<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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-pSB6A1-Prm/lac-GFP (N99)…sample with CI<br>
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-pSB6A1-PRM/lac-GFP (N99)…sample with CI<br>
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-pSB6A1--Prm/lac-GFP (JM2.300)…sample without CI<br>
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-pSB6A1--PRM/lac-GFP (JM2.300)…sample without CI<br>
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This N99 expresses CI from its genome constitutively.<br>
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This N99 strain expresses CI from its genome constitutively.<br>
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[[Image:titech2013_parts_K1139150_exp_Fig1.jpg|frameless|none|250px||]]
  
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[[Image:Titech2013_parts_K1139150_Fig1D.jpg|thumb|center|500px|]]
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<b>2. Assay protocol</b><br>
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1. Prepare overnight cultures of each cell at 37°C for 12 hours.<br>
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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>
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*We added IPTG in order to make sure to repress the expression of LacI derived from <i>E. coli</i> genome.<br>
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3. After 4 hours of induction, measure the fluorescence intensity with a flow cytometer.<br>
  
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'''1-2. Assay protocol'''<br>
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===Results===
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1. Prepare overnight culture of each cell at 37°C for 12 hours.<br>
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Fig. 1 shows the fluorescence intensity detected by flow cytometer.<br>  
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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>
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Fig. 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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We added IPTG in order to make sure to repress the expression of LacI derived from E. coli genome.<br>
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3. After 4 hours of induction, flow cytometer measurements for GFP expression.<br>
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===2. Results===
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[[Image:titech2013_parts_K1139150_Fig2.jpg|thumb|left|310px|<b>Fig. 1.</b> Fluorescence intensity detected by flow cytometer]]
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Fig. 2-1 shows the fluorescence intensity detected by flow cytometer.<br>  
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[[Image:titech2013_parts_K1139150_Fig3.jpg|thumb|none|240px|<b>Fig. 2.</b> Comparison of N99 and JM2.300]]
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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_Fig1B.jpg|thumb|center|500px|'''Fig. 2-1.''' Fluorescence intensity detected by flow cytometer]]
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<br>
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[[Image:Titech2013_parts_K1139150_Fig1C.jpg|thumb|center|500px|'''Fig. 2-2.''' Comparison of N99 and JM2.300]]
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===3. Discussion===
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===Discussion===
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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>
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N99 cells (CI+) showed higher fluorescence intensity than that of JM2.300 cells (CI-). From this result, we assume that our <i>RM/lac</i> hybrid promoter was actually activated by CI. In addition, N99 (IPTG-) showed lower fluorescence than that of N99 (IPTG+). From this result, we can assume that our <i>RM/lac</i> hybrid promoter was repressed by LacI derived from the <i>E. coli</i> genome.
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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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For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/RM-lac_Hybrid_Promoter_Assay our work in Tokyo_Tech 2013 wiki].
  
 
===Applications of BBa_K1139150===
 
===Applications of BBa_K1139150===

Latest revision as of 21:45, 28 October 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 strain expresses CI from its genome constitutively.

Titech2013 parts K1139150 exp Fig1.jpg

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).

Fig. 1. Fluorescence intensity detected by flow cytometer
Fig. 2. Comparison of N99 and JM2.300


Discussion

N99 cells (CI+) showed higher fluorescence intensity than that of JM2.300 cells (CI-). From this result, we assume that our RM/lac hybrid promoter was actually activated by CI. In addition, N99 (IPTG-) showed lower fluorescence than that of N99 (IPTG+). From this result, we can assume that our RM/lac hybrid promoter was repressed by LacI derived from the E. coli genome.

For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/RM-lac_Hybrid_Promoter_Assay our work in Tokyo_Tech 2013 wiki].

Applications of BBa_K1139150

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