Difference between revisions of "Part:BBa K1529797:Experience"
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+ | <partinfo>BBa_K1529265 short</partinfo> | ||
+ | __TOC__ | ||
+ | We confirmed whether the expression of CmR and C4HSL depends on the induction of 3OC12HSL. We inserted <i>lux</i> promoter (activated by 3OC12HSL-LuxR complex) upstream of <i>cmR</i> and <i>rhlI</i>, as an inducible promoter.<br> | ||
− | + | [[Image:Assay1_Flowchart.png|thumb|center|300px|<b>Fig. 1.</b> Flow chart of Assay1.]] | |
− | + | [[Image:Assay2_Flowchart.png|thumb|center|300px|<b>Fig. 2.</b> Flow chart of Assay2.]] | |
− | + | ||
− | + | アッセイの概要 | |
− | ===User Reviews | + | ==Materials and Methods== |
− | <!-- DON'T DELETE --><partinfo> | + | |
+ | ===Assay1 3OC12HSL-dependent Plux-CmR-RhlI cell growth assay=== | ||
+ | <b>1. Plasmid construction</b><br> | ||
+ | [[Image:Plasmid construction for assay1.png|thumb|right|300px|<b>Fig. 2.</b> Plasmid construction for assay1]] | ||
+ | Sample:<br> | ||
+ | pSB3K3-Plux-<i>cmR</i>-<i>rhlI</i> (<partinfo>BBa_K1529265</partinfo>)<br> | ||
+ | pSB6A1-Ptet-<i>luxR</i>-Plac-<i>rfp</i><br> | ||
+ | |||
+ | Positive control:<br> | ||
+ | pSB3K3-PlacIq-<i>cmR</i><br> | ||
+ | pSB6A1-Ptet-<i>luxR</i>-Plac-<i>rfp</i><br> | ||
+ | |||
+ | Negative control:<br> | ||
+ | pSB3K3-(Promoter less)-<i>cmR</i><br> | ||
+ | pSB6A1-Ptet-<i>luxR</i>-Plac-<i>rfp</i><br> | ||
+ | |||
+ | |||
+ | <b>2. Assay protocol</b><br> | ||
+ | *2-0 Strains<br> | ||
+ | DH5alpha (<i>E. coli</i> of high competence)<br> | ||
+ | JM2.300 (lacI22 <i>E. coli</i>)<br> | ||
+ | |||
+ | *2-1 Media<br> | ||
+ | Mix everything together in 1,000 mL autoclaved Elix H<sub>2</sub>O<br> | ||
+ | LB | ||
+ | {| class="wikitable" cellpadding="6" | ||
+ | |Bacto tryptone||10 g/L | ||
+ | |- | ||
+ | |Yeast Extract|| 5 g/L | ||
+ | |- | ||
+ | |NaCl||10 g/L | ||
+ | |} | ||
+ | |||
+ | *2-2 Others<br> | ||
+ | [ Antibiotics ]<br> | ||
+ | Ampisillin, Kanamycin, Chloramphenicol<br> | ||
+ | [ Inducer ]<br> | ||
+ | 3OC12HSL dissolved in DMSO (>100 µM)<br> | ||
+ | |||
+ | *2-3 Protocol<br> | ||
+ | [ Preparation ]<br> | ||
+ | 1.Transform JM2.300 with pSB3K3-Plux-M13-Plac-<i>GFP</i> and pSB6A1-Ptet-<i>luxR</i> <br> | ||
+ | 2.Grow overnight culture (Amp + Kan) of the transformed DH5alpha and JM109 at 37°C.<br> | ||
+ | 3.Take 30 µL of the overnight culture into 3 mL LB (Amp + Kan). (=> fresh culture)<br> | ||
+ | 4.Incubate the fresh culture at 37°C for 2 hours.<br> | ||
+ | 5.Add 3 µL of 5 µM 3OC6HSL in DMSO (final concentration: 5 nM) to the fresh culture and incubate at 37°C for 4 hours.<br> | ||
+ | 6.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.<br> | ||
+ | 7.Pipette the supernatant into a 1.5 mL tube.<br> | ||
+ | 8.Dilute it 100 times with water. (=> phage-particle-solution)<br> | ||
+ | |||
+ | [ Plaque formation ]<br> | ||
+ | 9.Transform JM109 with pSB6A1-Ptet-<i>luxR</i> <br> | ||
+ | 10.Grow overnight culture of the transformed JM109 at 37°C.<br> | ||
+ | 11.Melt YT soft agar using a microwave.<br> | ||
+ | 12.Add ampicillin to the YT soft agar.<br> | ||
+ | 13.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.<br> | ||
+ | 14.Dispense 400 µL of overnight culture of the transformed JM109 to a 1.5 mL tube.<br> | ||
+ | 15.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.<br> | ||
+ | 16.Transfer all the content of the 1.5 mL tube to the dispensed soft agar, mix them, and decant all of it on a YT plate.<br> | ||
+ | 17.Wait for YT soft agar to solidify at room temperature (for about 5 minutes).<br> | ||
+ | 18.Put an autoclaved piece of filter paper on the plate, and drip 20 mL of 3OC6HSL in DMSO (100 µM, 5 µM or DMSO only) on the piece of filter paper.<br> | ||
+ | |||
+ | ===Assay2 3OC12HSL-dependent C4HSL production assay=== | ||
+ | <b>1-1. Plasmid construction</b><br> | ||
+ | pSB3K3-Plux-<i>rmR<i/>-<i>rhlI</i> (<partinfo>BBa_K1529265</partinfo>)<br> | ||
+ | pSB6A1-Ptet-<i>luxR</i>-Plac-<i>rfp</i><br> | ||
+ | |||
+ | [[Image:titech2013_parts_K1139021_exp_Fig2.jpg|thumb|center|300px|<b>Fig. 2.</b> Plasmid construction for assay]] | ||
+ | |||
+ | <b>1-2. Assay protocol</b><br> | ||
+ | *2-0 Strains<br> | ||
+ | JM2.300 <br> | ||
+ | |||
+ | *2-1 Media<br> | ||
+ | Mix everything together in 1,000 mL autoclaved Elix H<sub>2</sub>O<br> | ||
+ | LB | ||
+ | {| class="wikitable" cellpadding="6" | ||
+ | |Bacto tryptone||10 g/L | ||
+ | |- | ||
+ | |Yeast Extract|| 5 g/L | ||
+ | |- | ||
+ | |NaCl||10 g/L | ||
+ | |} | ||
+ | |||
+ | *2-2 Others<br> | ||
+ | 3OC12HSL dissolved in DMSO (>100 µM)<br> | ||
+ | Autoclaved pieces of filter paper (about 1.5 cm in diameter) <br> | ||
+ | |||
+ | *2-3 Protocol<br> | ||
+ | [ Preparation ]<br> | ||
+ | 1.Transform DH5alpha with pSB3K3-Plux-M13-Plac-<i>GFP</i> and pSB6A1-Ptet-<i>luxR</i> <br> | ||
+ | 2.Grow overnight culture (Amp + Kan) of the transformed DH5alpha and JM109 at 37°C.<br> | ||
+ | 3.Take 30 µL of the overnight culture into 3 mL LB (Amp + Kan). (=> fresh culture)<br> | ||
+ | 4.Incubate the fresh culture at 37°C for 2 hours.<br> | ||
+ | 5.Add 3 µL of 5 µM 3OC6HSL in DMSO (final concentration: 5 nM) to the fresh culture and incubate at 37°C for 4 hours.<br> | ||
+ | 6.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.<br> | ||
+ | 7.Pipette the supernatant into a 1.5 mL tube.<br> | ||
+ | 8.Dilute it 100 times with water. (=> phage-particle-solution)<br> | ||
+ | |||
+ | [ Plaque formation ]<br> | ||
+ | 9.Transform JM109 with pSB6A1-Ptet-<i>luxR</i> <br> | ||
+ | 10.Grow overnight culture of the transformed JM109 at 37°C.<br> | ||
+ | 11.Melt YT soft agar using a microwave.<br> | ||
+ | 12.Add ampicillin to the YT soft agar.<br> | ||
+ | 13.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.<br> | ||
+ | 14.Dispense 400 µL of overnight culture of the transformed JM109 to a 1.5 mL tube.<br> | ||
+ | 15.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.<br> | ||
+ | 16.Transfer all the content of the 1.5 mL tube to the dispensed soft agar, mix them, and decant all of it on a YT plate.<br> | ||
+ | 17.Wait for YT soft agar to solidify at room temperature (for about 5 minutes).<br> | ||
+ | 18.Put an autoclaved piece of filter paper on the plate, and drip 20 mL of 3OC6HSL in DMSO (100 µM, 5 µM or DMSO only) on the piece of filter paper.<br> | ||
+ | |||
+ | ==Result== | ||
+ | ===Result1 3OC12HSL-dependent Plux-CmR-RhlI cell growth assay=== | ||
+ | |||
+ | |||
+ | |||
+ | ===Result2 3OC12HSL-dependent C4HSL production assay=== | ||
+ | |||
+ | |||
+ | For more information, see [http://2014.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2013 wiki]. | ||
+ | |||
+ | ==Applications of BBa_K1529265== | ||
+ | |||
+ | ==User Reviews== | ||
+ | <!-- DON'T DELETE --><partinfo>BBa_K1529265 StartReviews</partinfo> | ||
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− | <partinfo> | + | <partinfo>BBa_K1529265 AddReview number</partinfo> |
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Revision as of 05:25, 8 October 2014
Plux_CmR_RhlI
We confirmed whether the expression of CmR and C4HSL depends on the induction of 3OC12HSL. We inserted lux promoter (activated by 3OC12HSL-LuxR complex) upstream of cmR and rhlI, as an inducible promoter.
アッセイの概要
Materials and Methods
Assay1 3OC12HSL-dependent Plux-CmR-RhlI cell growth assay
1. Plasmid construction
Sample:
pSB3K3-Plux-cmR-rhlI (BBa_K1529265)
pSB6A1-Ptet-luxR-Plac-rfp
Positive control:
pSB3K3-PlacIq-cmR
pSB6A1-Ptet-luxR-Plac-rfp
Negative control:
pSB3K3-(Promoter less)-cmR
pSB6A1-Ptet-luxR-Plac-rfp
2. Assay protocol
- 2-0 Strains
DH5alpha (E. coli of high competence)
JM2.300 (lacI22 E. coli)
- 2-1 Media
Mix everything together in 1,000 mL autoclaved Elix H2O
LB
Bacto tryptone | 10 g/L |
Yeast Extract | 5 g/L |
NaCl | 10 g/L |
- 2-2 Others
[ Antibiotics ]
Ampisillin, Kanamycin, Chloramphenicol
[ Inducer ]
3OC12HSL dissolved in DMSO (>100 µM)
- 2-3 Protocol
[ Preparation ]
1.Transform JM2.300 with pSB3K3-Plux-M13-Plac-GFP and pSB6A1-Ptet-luxR
2.Grow overnight culture (Amp + Kan) of the transformed DH5alpha and JM109 at 37°C.
3.Take 30 µL of the overnight culture into 3 mL LB (Amp + Kan). (=> fresh culture)
4.Incubate the fresh culture at 37°C for 2 hours.
5.Add 3 µL of 5 µM 3OC6HSL in DMSO (final concentration: 5 nM) to the fresh culture and incubate at 37°C for 4 hours.
6.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.
7.Pipette the supernatant into a 1.5 mL tube.
8.Dilute it 100 times with water. (=> phage-particle-solution)
[ Plaque formation ]
9.Transform JM109 with pSB6A1-Ptet-luxR
10.Grow overnight culture of the transformed JM109 at 37°C.
11.Melt YT soft agar using a microwave.
12.Add ampicillin to the YT soft agar.
13.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.
14.Dispense 400 µL of overnight culture of the transformed JM109 to a 1.5 mL tube.
15.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.
16.Transfer all the content of the 1.5 mL tube to the dispensed soft agar, mix them, and decant all of it on a YT plate.
17.Wait for YT soft agar to solidify at room temperature (for about 5 minutes).
18.Put an autoclaved piece of filter paper on the plate, and drip 20 mL of 3OC6HSL in DMSO (100 µM, 5 µM or DMSO only) on the piece of filter paper.
Assay2 3OC12HSL-dependent C4HSL production assay
1-1. Plasmid construction
pSB3K3-Plux-rmR<i/>-<i>rhlI (BBa_K1529265)
pSB6A1-Ptet-luxR-Plac-rfp
1-2. Assay protocol
- 2-0 Strains
JM2.300
- 2-1 Media
Mix everything together in 1,000 mL autoclaved Elix H2O
LB
Bacto tryptone | 10 g/L |
Yeast Extract | 5 g/L |
NaCl | 10 g/L |
- 2-2 Others
3OC12HSL dissolved in DMSO (>100 µM)
Autoclaved pieces of filter paper (about 1.5 cm in diameter)
- 2-3 Protocol
[ Preparation ]
1.Transform DH5alpha with pSB3K3-Plux-M13-Plac-GFP and pSB6A1-Ptet-luxR
2.Grow overnight culture (Amp + Kan) of the transformed DH5alpha and JM109 at 37°C.
3.Take 30 µL of the overnight culture into 3 mL LB (Amp + Kan). (=> fresh culture)
4.Incubate the fresh culture at 37°C for 2 hours.
5.Add 3 µL of 5 µM 3OC6HSL in DMSO (final concentration: 5 nM) to the fresh culture and incubate at 37°C for 4 hours.
6.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.
7.Pipette the supernatant into a 1.5 mL tube.
8.Dilute it 100 times with water. (=> phage-particle-solution)
[ Plaque formation ]
9.Transform JM109 with pSB6A1-Ptet-luxR
10.Grow overnight culture of the transformed JM109 at 37°C.
11.Melt YT soft agar using a microwave.
12.Add ampicillin to the YT soft agar.
13.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.
14.Dispense 400 µL of overnight culture of the transformed JM109 to a 1.5 mL tube.
15.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.
16.Transfer all the content of the 1.5 mL tube to the dispensed soft agar, mix them, and decant all of it on a YT plate.
17.Wait for YT soft agar to solidify at room temperature (for about 5 minutes).
18.Put an autoclaved piece of filter paper on the plate, and drip 20 mL of 3OC6HSL in DMSO (100 µM, 5 µM or DMSO only) on the piece of filter paper.
Result
Result1 3OC12HSL-dependent Plux-CmR-RhlI cell growth assay
Result2 3OC12HSL-dependent C4HSL production assay
For more information, see [http://2014.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2013 wiki].
Applications of BBa_K1529265
User Reviews
UNIQ5054b8ffdbeb08d1-partinfo-00000003-QINU UNIQ5054b8ffdbeb08d1-partinfo-00000004-QINU