Difference between revisions of "Part:BBa K1139020:Experience"

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__NOTOC__
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<partinfo>BBa_K1139020 short</partinfo>
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<partinfo>BBa_K1139020</partinfo>
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__TOC__
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===Materials and Methods===
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===1. Materials and Method===
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<b>1. Plasmid construction</b><br>
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'''1-0 Plasmid construction'''<br>
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pSB3K3- PlacI<sup>q</sup>-M13-Plac-GFP (<partinfo>BBa_K1139020</partinfo>)<br>
 
pSB3K3- PlacI<sup>q</sup>-M13-Plac-GFP (<partinfo>BBa_K1139020</partinfo>)<br>
 
pSB3K3-M13-Plac-GFP (<partinfo>BBa_K1139022</partinfo>)<br>
 
pSB3K3-M13-Plac-GFP (<partinfo>BBa_K1139022</partinfo>)<br>
  
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[[Image:Titech2013_parts_K1139020_EX_Fig1.jpg|thumb|left|500px|'''Fig. 1.''' Plasmid construction for assay]]
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[[Image:titech2013_parts_K1139020_exp_Fig1.png|thumb|left|240px|<b>Fig. 1.</b> Plasmid construction for assay]]
  
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
  
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'''1-1 Strain'''<br>
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<b>2. Strain</b><br>
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DH5alpha (''Ecoli'' of high competence)<br>
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DH5alpha (<i>Ecoli</i> of high competence)<br>
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JM109 (F+ strain ''Ecoli'')<br>
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JM109 (F+ strain <i>Ecoli</i>)<br>
  
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'''1-2 Media'''<br>
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<b>3. Media</b><br>
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Mix everything together in 1000 mL autoclaved Elix H<small>2</small>O<br>
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Mix everything together in 1,000 mL autoclaved Elix H<sub>2</sub>O<br>
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LB<br>
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LB
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{| class="wikitable" cellpadding="6"
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|Bacto tryptone||10 g/L
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|-
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|Yeast Extract||&nbsp;&nbsp;5 g/L
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|-
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|NaCl||10 g/L
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|}
  
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[[Image:Titech2013_parts_K1139020_EX_Tab1.jpg|frameless|left|500px|]]
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YT plate
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{| class="wikitable" cellpadding="6"
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|Bacto tryptone||&nbsp;&nbsp;8 g/L
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|-
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|Yeast Extract||&nbsp;&nbsp;5 g/L
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|-
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|NaCl||&nbsp;&nbsp;5 g/L
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|-
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|Agarose||15 g/L
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|}
  
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<br><br><br><br>
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YT soft agar
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{| class="wikitable" cellpadding="6"
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|Bacto tryptone||8 g/L
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|-
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|Yeast Extract||5 g/L
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|-
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|NaCl||5 g/L
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|-
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|Agarose||6 g/L
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|}
  
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YT soft agar<br>
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<b>4. Protocol</b><br>
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[[Image:Titech2013_parts_K1139020_EX_Tab2.jpg|frameless|left|500px|]]
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<br><br><br><br>
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YT plate<br>
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[[Image:Titech2013_parts_K1139020_EX_Tab3.jpg|frameless|left|500px|]]
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<br><br><br><br>
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'''1-3Protocol'''<br>
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*Preparation<br>
 
*Preparation<br>
 
1.Transform DH5alpha with pSB3K3- PlacI<sup>q</sup>-M13.<br>
 
1.Transform DH5alpha with pSB3K3- PlacI<sup>q</sup>-M13.<br>
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3.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.<br>
 
3.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.<br>
 
4.Pipette the supernatant into a 1.5 mL tube.<br>
 
4.Pipette the supernatant into a 1.5 mL tube.<br>
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5.Dilute it 100 times with water.  (=>phage-particle-solution)<br>
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5.Dilute it 100 times with water.  (=> phage-particle-solution)<br>
 
6.Melt YT soft agar using a microwave.<br>
 
6.Melt YT soft agar using a microwave.<br>
 
7.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.<br>
 
7.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.<br>
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10.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>
 
10.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>
  
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===2. Result===
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===Result===
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The result of the plaque forming assay is showed in Fig. 2.  M13 phage genes and M13 origin worked together with ''lacI<sup>q</sup>'' promoter and pSB origin.<br>
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The result of the plaque forming assay is showed in Fig. 2-A and 2-B.  M13 phage genes and M13 origin worked together with <i>lacI<sup>q</sup></i> promoter and pSB origin.<br>
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[[Image:titech2013_parts_K1139020_exp_Fig1.jpg|thumb|left|400px|<b>Fig. 2-A.</b> The plaques]]
  
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[[Image:Titech2013_parts_K1139020_Fig1.jpg|thumb|left|500px|'''Fig. 2-A.''' The plaques]]
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[[Image:titech2013_parts_K1139020_exp_Fig2.jpg|thumb|left|400px|<b>Fig. 2-B.</b>  The negative control]]
  
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[[Image:Titech2013_parts_K1139020_Fig2.jpg|thumb|left|500px|'''Fig. 2-B.'''  The negative control]]
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
  
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<br><br><br><br><br><br><br><br><br><br><br><br>
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<b>Fig. 2-A.</b><br>
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The plaques were formed using JM109 overnight culture and phage-particle-solution.  The phage particles were obtained from the supernatant of the overnight culture of transformed DH5alpha (with pSB3K3- PlacI<sup>q</sup>-M13) at 9,000g.  Mixture of 3.5 mL YT soft agar, 100 µL of X100 supernatant and 400 µL of overnight culture of JM109 was poured on a YT plate.<br>
  
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'''Fig. 2-A.'''<br>
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<b>Fig. 2-B.</b><br>
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The plaques were formed using JM109 overnight culture and phage-particle-solution.  The phage particles were obtained from the supernatant of the overnight culture of transformed DH5alpha (with pSB3K3- PlacI<sup>q</sup>-M13) at 9,000g.  Mixture of 3.5 mL YT soft agar, 100 µL of X 100 supernatant and 400 µL of overnight culture of JM109 was poured on a YT plate.<br>
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A mixture of only 3.5 mL YT soft agar and 500 µL of overnight culture of JM109 was poured on a YT plate.<br>
  
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'''Fig. 2-B.'''<br>
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<br>
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A mixture of only 3.5 mL YT soft agar and 500 microL of overnight culture of JM109 was poured on a YT plate.<br>
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For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/pSB-M13_Plasmid_Assay our work in Tokyo_Tech 2013 wiki].
  
 
===Applications of BBa_K1139020===
 
===Applications of BBa_K1139020===

Latest revision as of 02:50, 27 September 2013

PlacIq-M13-Plac-GFP on pSB3

Materials and Methods

1. Plasmid construction
pSB3K3- PlacIq-M13-Plac-GFP (BBa_K1139020)
pSB3K3-M13-Plac-GFP (BBa_K1139022)

Fig. 1. Plasmid construction for assay

















2. Strain
DH5alpha (Ecoli of high competence)
JM109 (F+ strain Ecoli)

3. 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

YT plate

Bacto tryptone   8 g/L
Yeast Extract   5 g/L
NaCl   5 g/L
Agarose 15 g/L

YT soft agar

Bacto tryptone 8 g/L
Yeast Extract 5 g/L
NaCl 5 g/L
Agarose 6 g/L

4. Protocol

  • Preparation

1.Transform DH5alpha with pSB3K3- PlacIq-M13.
2.Grow overnight culture of the transformed DH5alpha and JM109 at 37°C.

  • Plaque formation

3.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.
4.Pipette the supernatant into a 1.5 mL tube.
5.Dilute it 100 times with water. (=> phage-particle-solution)
6.Melt YT soft agar using a microwave.
7.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.
8.Dispense 400 µL of overnight culture of JM109 to a 1.5 mL tube.
9.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.
10.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.

Result

The result of the plaque forming assay is showed in Fig. 2-A and 2-B. M13 phage genes and M13 origin worked together with lacIq promoter and pSB origin.

Fig. 2-A. The plaques
Fig. 2-B. The negative control


















Fig. 2-A.
The plaques were formed using JM109 overnight culture and phage-particle-solution. The phage particles were obtained from the supernatant of the overnight culture of transformed DH5alpha (with pSB3K3- PlacIq-M13) at 9,000g. Mixture of 3.5 mL YT soft agar, 100 µL of X100 supernatant and 400 µL of overnight culture of JM109 was poured on a YT plate.

Fig. 2-B.
A mixture of only 3.5 mL YT soft agar and 500 µL of overnight culture of JM109 was poured on a YT plate.


For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/pSB-M13_Plasmid_Assay our work in Tokyo_Tech 2013 wiki].

Applications of BBa_K1139020

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