Team:NYMU-Taipei/Experiment/Wet Lab

From 2013.igem.org

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{{:Team:NYMU-Taipei/Header}}
{{:Team:NYMU-Taipei/Header}}
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=N. ceranae experiment protocols=
 
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==Extraction of N. ceranae Spores from Bees==
 
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[[File:Extraction.png|frame|center]]
 
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==Purification of N. ceranae Spores==
 
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[[File:Nymu-Purification.png|frame|center]]
 
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==N. ceranae Genomic DNA Extraction==
 
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[[File:Nymu-Genomic.png|frame|center]]
 
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==Germination of N. ceranae Spores==
 
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[[File:Nymu-Germination.PNG|frame|center]]
 
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=Coding Sequence Testing=
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== Data for Our Favorite Parts ==
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We define the germination degree as follows:
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# An '''OxyR-induced Promoter'''  [http://parts.igem.org/Part:BBa_K1104201  BBa_K1104201] (TrxC promoter)
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# A '''Transcription Factor Activated by Oxidative Stress''' [http://parts.igem.org/Part:BBa_K1104200 BBa_K1104200] (OxyR) - We apply a constitutive promoter to the upstream region of OxyR coding sequence to overexpress the OxyR protein, which is expected to boost the sensitivity of our ROS-induced promotors.
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# An '''antimicrobial peptide''' [http://parts.igem.org/Part:BBa_K1104301 BBa_K1104200 ] (Defensin1) - The mechanism which insects use to protect against the invasion of microbes.
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== Data for Pre-Existing and Optimized Parts ==
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=== AhpCp ===
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We successfully improved the biobrick part: ahpC promoter [http://parts.igem.org/Part:BBa_K362001 ahpC (K362001)]. Originally created by the KIT-Kyoto 2010 iGEM team by taking the upstream 1000bp of the AhpC gene, we improved the promoter by first mutating the PstI cutting site to make it conform to Assembly standard 10. We then designed specific primers to cut short the original sequence of AhpCp promoter to trim out unneeded sequences make it more efficient. More details about the design can be found on the [[Team:NYMU-Taipei/Project/Inhibition/Sensor|Nosema Sensor]] page.
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{|
{|
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|-
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|[[File:NYMU_Amutate1000.png|thumb|600px|center|'''AhpCp1000[http://parts.igem.org/Part:BBa_K1104203 Part:BBa_K1104203]'''<br>We succesfully mutated the Pst1 site (ctgcag->ctacag) of K362001.]]
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|[[File:Nymu-Ptps5 .png|thumb|250px|''' 1 point:A spore begins its budding reproduction.''']]||[[File:POINT 1.png|thumb|250px|''' 2 points:A new spore is ccompletely germinanated from the old one.''']]
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|The supplementary characterization and the application of AhpC promoter has been added to the experience page of [http://parts.igem.org/Part:BBa_K1104203 BBa_K1104203 (AhpCp1000)].
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|}
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The AhpCp1000 derived OxyR-induced promoters we created are as follows:
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{|
{|
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|-
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|[[File:NYMU_AhpC2D1.png|thumb|400px|center|'''AhpCp2D1([http://parts.igem.org/Part:BBa_K1104204 Part:BBa_K1104204])'''<br>We strimmed DsbG coding out of the original sequence, making the functional sequence shorter and more efficient.]]
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|[[File:Nymu-POINT 2.png|thumb|250px|''' 3 points:A cluster of duplicated spores is formed.''']]||[[File:Nymu-Tv10 ptp unsure.PNG|thumb|250px|''' 4 points:A Spore extrude a polar filament.''']]
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|[http://parts.igem.org/Part:BBa_K1104204 BBa_K1104204] (AhpCp2D1 promoter)Composed of '''AhpCp2''', '''DsbGp''', and '''AhpCp1'''. DsbG coding sequence of AhpCp1000 was trimmed out.
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|}
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{|
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|[[File:NYMU_AhpCD1.png|thumb|400px|center|'''AhpCpD1''']]
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|[http://parts.igem.org/Part:BBa_K1104206 BBa_K1104206] (AhpCpD1 promoter)This is a '''bidirectional design''', Composed of '''DsbGp(reverse)''' and '''AhpCp1(forward)''', and a shared TFBS for OxyR.
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|}
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{|
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|[[File:NYMU_AhpC2.png|thumb|200px|center|'''AhpCp2'''<br>]]
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|[http://parts.igem.org/Part:BBa_K1104205 BBa_K1104205] (AhpCp2 promoter)                                                                               
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|}
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{|
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|[[File:NYMU_AhpC1.png|thumb|200px|center|'''AhpCp1''']]
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|[http://parts.igem.org/Part:BBa_K1104207 BBa_K1104207] (AhpCp1 promoter)                                                           
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|}
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{|
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|[[File:NYMU_DsbG.png|thumb|center|400px|'''DsbGp''']]
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|[http://parts.igem.org/Part:BBa_K1104207 BBa_K1104208] (DsbGp promoter)                                                                                   
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|}
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=== SufAp ===
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We also characterized the pre-existing SufAp promoter ([http://parts.igem.org/Part:K362005 BBa_K362005]) under different H<sub>2</sub>0<sub>2</sub> concentrations.
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== List of Our Parts ==
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<groupparts>iGEM013 NYMU-Taipei</groupparts>
{{:Team:NYMU-Taipei/Footer}}
{{:Team:NYMU-Taipei/Footer}}

Latest revision as of 17:23, 28 October 2013

National Yang Ming University


Contents

Data for Our Favorite Parts

  1. An OxyR-induced Promoter [http://parts.igem.org/Part:BBa_K1104201 BBa_K1104201] (TrxC promoter)
  2. A Transcription Factor Activated by Oxidative Stress [http://parts.igem.org/Part:BBa_K1104200 BBa_K1104200] (OxyR) - We apply a constitutive promoter to the upstream region of OxyR coding sequence to overexpress the OxyR protein, which is expected to boost the sensitivity of our ROS-induced promotors.
  3. An antimicrobial peptide [http://parts.igem.org/Part:BBa_K1104301 BBa_K1104200 ] (Defensin1) - The mechanism which insects use to protect against the invasion of microbes.


Data for Pre-Existing and Optimized Parts

AhpCp

We successfully improved the biobrick part: ahpC promoter [http://parts.igem.org/Part:BBa_K362001 ahpC (K362001)]. Originally created by the KIT-Kyoto 2010 iGEM team by taking the upstream 1000bp of the AhpC gene, we improved the promoter by first mutating the PstI cutting site to make it conform to Assembly standard 10. We then designed specific primers to cut short the original sequence of AhpCp promoter to trim out unneeded sequences make it more efficient. More details about the design can be found on the Nosema Sensor page.

AhpCp1000[http://parts.igem.org/Part:BBa_K1104203 Part:BBa_K1104203]
We succesfully mutated the Pst1 site (ctgcag->ctacag) of K362001.
The supplementary characterization and the application of AhpC promoter has been added to the experience page of [http://parts.igem.org/Part:BBa_K1104203 BBa_K1104203 (AhpCp1000)].

The AhpCp1000 derived OxyR-induced promoters we created are as follows:

AhpCp2D1([http://parts.igem.org/Part:BBa_K1104204 Part:BBa_K1104204])
We strimmed DsbG coding out of the original sequence, making the functional sequence shorter and more efficient.
[http://parts.igem.org/Part:BBa_K1104204 BBa_K1104204] (AhpCp2D1 promoter)Composed of AhpCp2, DsbGp, and AhpCp1. DsbG coding sequence of AhpCp1000 was trimmed out.
AhpCpD1
[http://parts.igem.org/Part:BBa_K1104206 BBa_K1104206] (AhpCpD1 promoter)This is a bidirectional design, Composed of DsbGp(reverse) and AhpCp1(forward), and a shared TFBS for OxyR.
AhpCp2
[http://parts.igem.org/Part:BBa_K1104205 BBa_K1104205] (AhpCp2 promoter)
AhpCp1
[http://parts.igem.org/Part:BBa_K1104207 BBa_K1104207] (AhpCp1 promoter)
DsbGp
[http://parts.igem.org/Part:BBa_K1104207 BBa_K1104208] (DsbGp promoter)

SufAp

We also characterized the pre-existing SufAp promoter ([http://parts.igem.org/Part:K362005 BBa_K362005]) under different H202 concentrations.

List of Our Parts

<groupparts>iGEM013 NYMU-Taipei</groupparts>


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