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| <partinfo>BBa_K1529265 short</partinfo> | | <partinfo>BBa_K1529265 short</partinfo> |
| __TOC__ | | __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>
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− | [[Image:Assay1_Flowchart.png|thumb|center|300px|<b>Fig. 1.</b> Flow chart of Assay1.]]
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− | [[Image:Assay2_Flowchart.png|thumb|center|300px|<b>Fig. 2.</b> Flow chart of Assay2.]]
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| | | |
− | アッセイの概要
| + | ==Materials and Methods== |
− | | + | |
− | ==3OC12HSL-dependent CmR expression== | + | |
− | | + | |
− | ===3OC12HSL-dependent CmR expression Result===
| + | |
− | <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>
| + | |
| | | |
| ===3OC12HSL-dependent CmR expression Protocol=== | | ===3OC12HSL-dependent CmR expression Protocol=== |
− | <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>
| + | ===3OC12HSL-dependent C4HSL production Protocol=== |
− | *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>
| + | ==Results== |
− | 3OC12HSL dissolved in DMSO (>100 µM)<br>
| + | |
− | Autoclaved pieces of filter paper (about 1.5 cm in diameter) <br>
| + | |
| | | |
− | *2-3 Protocol<br>
| + | ===3OC12HSL-dependent CmR expression Result=== |
− | [ 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>
| + | |
− | | + | |
− | ==3OC12HSL-dependent C4HSL expression==
| + | |
− | ===3OC12HSL-dependent C4HSL expression=== | + | |
| | | |
| | | |
| | | |
− | ===3OC12HSL-dependent C4HSL expression=== | + | ===3OC12HSL-dependent C4HSL production Result=== |
| | | |
| | | |
− | For more information, see [http://2014.igem.org/Team:Tokyo_Tech our work in Tokyo_Tech 2013 wiki]. | + | For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/3OC12HSL-dependent_C4HSL_production our work in Tokyo_Tech 2014 wiki]. |
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− | ==Applications of BBa_K1529265== | + | ==Applications of BBa_K1529797== |
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| ==User Reviews== | | ==User Reviews== |
− | <!-- DON'T DELETE --><partinfo>BBa_K1529265 StartReviews</partinfo> | + | <!-- DON'T DELETE --><partinfo>BBa_K1529797 StartReviews</partinfo> |
| <!-- Template for a user review | | <!-- Template for a user review |
| {|width='80%' style='border:1px solid gray' | | {|width='80%' style='border:1px solid gray' |
| |- | | |- |
| |width='10%'| | | |width='10%'| |
− | <partinfo>BBa_K1529265 AddReview number</partinfo> | + | <partinfo>BBa_K1529797 AddReview number</partinfo> |
| <I>Username</I> | | <I>Username</I> |
| |width='60%' valign='top'| | | |width='60%' valign='top'| |
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| |}; | | |}; |
| <!-- End of the user review template --> | | <!-- End of the user review template --> |
− | <!-- DON'T DELETE --><partinfo>BBa_K1529265 EndReviews</partinfo> | + | <!-- DON'T DELETE --><partinfo>BBa_K1529797 EndReviews</partinfo> |
For more information, see [http://2014.igem.org/Team:Tokyo_Tech/Experiment/3OC12HSL-dependent_C4HSL_production our work in Tokyo_Tech 2014 wiki].
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