Difference between revisions of "Part:BBa K1613012"
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There is a QsrR Binding Site between promoter and RBS. Which can combine with Quinone-sensing and response Repressor(QsrR). And inhibit the transcription. While QsrR combines with BaP-1,6-Quinone, the QsrR will be activated and depart the Binding Site. That makes the transcription of downstream.
 
There is a QsrR Binding Site between promoter and RBS. Which can combine with Quinone-sensing and response Repressor(QsrR). And inhibit the transcription. While QsrR combines with BaP-1,6-Quinone, the QsrR will be activated and depart the Binding Site. That makes the transcription of downstream.
  
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<p style="font-size:120%">'''Gel Electrophoresis'''</p>
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<p style="font-size:150%">'''Gel Electrophoresis'''</p>
  
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[[File:HSNU-TAIPEI-BBa_K1613012.png]]
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[[File:HSNU-TAIPEI-BBa_K1613012.png | 400px | thumb | Fig.1:The Gel Electrophoresis picture of BBa_K1613012. The plasmid was cut by restriction enzymes EcoRI and SpeI. There are three bands in the picture: Plasmids, Backbones, and Parts.]]
  
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Fig.1:The Gel Electrophoresis picture of BBa_K1613012. The plasmid was cut by restriction enzymes EcoRI and SpeI. There are three bands in the picture: Plasmids, Backbones, and Parts.
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=== E.Coli Survival ===
 +
 
 +
To ensure the biosensor can work in the toxin solution. We did experiment of E.coli survival. To make sure the toxin won't kill it.
 +
 
 +
=== Procedure ===
 +
First we culture DH5α with LB only plate for 15hr. Then,pick one colony in the LB broth,and liquid culture for 15hr.
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Take 80μL into 2ml LB broth × 6 tubes and then culture 1 hr.
 +
After 1hr,add 20μL benzo[a]pryene into three tubes(conc. Is 2000ppb(A thousand times the standard value))
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And add 20μL DMSO into the other tubes.Then,culture for 3hr.
 +
After 3hr,dilute the broth to 10-6
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And take 200μL to spread the plate.
 +
 
 +
=== Results ===
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{| class="wikitable" style="width: 400px; height: 200px; text-align: center;"
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|+ Table1: E. coli on the agar plate.
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! colspan="4" | Influence of benzo[a]pyrene on E.coli(DH5&alpha;) survival rate
 +
|-
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! scope="col" | Group
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! scope="col" colspan="3" | Colony
 +
|-
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! scope="row" | Benzo[A]Pyrene
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| 237
 +
| 201
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| 104
 +
|-
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! scope="row" | Control
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| 189
 +
| 51
 +
| 233
 +
|}
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[[File:HSNU-TAIPEI-BZP-904-2.jpg | left | 400px | thumb | Fig2: Benzo[a]pyrene Group]]
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<br clear="both">
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[[File:HSNU-TAIPEI-BZP-904-3.jpg | left | 400px | thumb | Fig3:Control Group]]
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<br clear="both">
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 +
There is no difference in amount of E.coli between two groups which means benzopyrene will not affect the survival of E.coli.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1613012 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1613013 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1613012 parameters</partinfo>
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<partinfo>BBa_K1613013 parameters</partinfo>
 
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Latest revision as of 20:32, 26 September 2015

QsrR Binding Site inhibits the transcription and translation of Red Fluorescent Protein.

There is a QsrR Binding Site between promoter and RBS. Which can combine with Quinone-sensing and response Repressor(QsrR). And inhibit the transcription. While QsrR combines with BaP-1,6-Quinone, the QsrR will be activated and depart the Binding Site. That makes the transcription of downstream.

Gel Electrophoresis

Fig.1:The Gel Electrophoresis picture of BBa_K1613012. The plasmid was cut by restriction enzymes EcoRI and SpeI. There are three bands in the picture: Plasmids, Backbones, and Parts.


E.Coli Survival

To ensure the biosensor can work in the toxin solution. We did experiment of E.coli survival. To make sure the toxin won't kill it.

Procedure

First we culture DH5α with LB only plate for 15hr. Then,pick one colony in the LB broth,and liquid culture for 15hr. Take 80μL into 2ml LB broth × 6 tubes and then culture 1 hr. After 1hr,add 20μL benzo[a]pryene into three tubes(conc. Is 2000ppb(A thousand times the standard value)) And add 20μL DMSO into the other tubes.Then,culture for 3hr. After 3hr,dilute the broth to 10-6 And take 200μL to spread the plate.

Results

Table1: E. coli on the agar plate.
Influence of benzo[a]pyrene on E.coli(DH5α) survival rate
Group Colony
Benzo[A]Pyrene 237 201 104
Control 189 51 233
Fig2: Benzo[a]pyrene Group


Fig3:Control Group


There is no difference in amount of E.coli between two groups which means benzopyrene will not affect the survival of E.coli.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1409
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1570