Difference between revisions of "Part:BBa K929302"
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<partinfo>BBa_K929302 short</partinfo>
 
<partinfo>BBa_K929302 short</partinfo>
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==='''AID in Potsdam Standard'''===
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<br><br>
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<p style="background-color: rgb(238, 221, 130); font-weight: bold;">Introduction</p>
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{| style="color:black" cellpadding="6" cellspacing="1" border="2" align="right"
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! colspan="2" style="background:rgb(240, 20, 70);"|[https://parts.igem.org/Part:BBa_K929302 AID in Potsdam Standard]
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|-
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! colspan="2"|[[Image:UP12_aid_in_Potsdam_Standard.png|300px]]
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|-
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|'''BioBrick Nr.'''
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|[https://parts.igem.org/Part:BBa_K929302 BBa_K929302]
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|-
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|'''RFC standard'''
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|[http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard RFC 10], [http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard Potsdam Standard]
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|-
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|'''Requirement'''
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|pSB1C3<br>
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|-
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|'''Source'''
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|existing part:([https://parts.igem.org/Part:BBa_K103001 BBa_K103001])
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|-
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|'''Submitted by'''
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|[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]
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|}
  
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<p style="background-color: rgb(238, 221, 130); font-weight: bold;">Introduction</p>
 
 
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AID is known to be responsible for somatic hypermutation and the class-switch recombination of immunoglobulin in B cells. This enzyme of 28 kDa originally occurs in B cells but does also show activity after transfection into CHO cells. AID induces the deamination of cytidine to uridine at actively transcribed single strand DNA. The replacement of cytidine by uridine leads to a mismatch during DNA replication and integrates a single base substitution predominantly in the immunoglobulin genes.<br>
 
AID is known to be responsible for somatic hypermutation and the class-switch recombination of immunoglobulin in B cells. This enzyme of 28 kDa originally occurs in B cells but does also show activity after transfection into CHO cells. AID induces the deamination of cytidine to uridine at actively transcribed single strand DNA. The replacement of cytidine by uridine leads to a mismatch during DNA replication and integrates a single base substitution predominantly in the immunoglobulin genes.<br>
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<p style="background-color: rgb(238, 221, 130); font-weight: bold;">Cloning with Potsdam Standard</p>
 
<p style="background-color: rgb(238, 221, 130); font-weight: bold;">Cloning with Potsdam Standard</p>
 
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To generate this part, we used the [http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard Potsdam Standard] for cloning the AID into the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K929301 Potsdam Standard Backbone]. Here, we tested four different ligation conditions: 1. with electrophoretic unpurified, digested Potsdam Standard backbone with and without ligase and 2. with electrophoretic purified digested Potsdam Standard backbone with and without ligase. The results are summarized in fig. 1. We can see that with ligase the ligation success is much higher than without, and that a electrophoretic purification of the backbone improve the ligation efficiency.
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To generate this part, we used the [http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard Potsdam Standard] for cloning the AID into the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K929301 Potsdam Standard Backbone]. Here, we tested four different ligation conditions: 1. with electrophoretic unpurified, digested Potsdam Standard backbone with and without ligase and 2. with electrophoretic purified digested Potsdam Standard backbone with and without ligase. To compute the efficiency, we counted the colonies without and with red fluorescence indicating a failed ligation reaction (fig. 1). The results are summarized in fig. 2. We can see that with ligase the ligation success is much higher than without, and that a electrophoretic purification of the backbone improve the ligation efficiency.
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[[image:UP_12_Potstdam_Standard.png|400px|center|thumb|Fig. 1: Relative ligation success of the four conditions]]
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[[image:UP12_colonies.png|center|300px|thumb|Fig. 1: Result of the transformation in ''E. coli'']]
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[[image:UP_12_Potstdam_Standard.png|400px|center|thumb|Fig. 2: Relative ligation success of the four conditions]]
 
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Latest revision as of 14:07, 28 September 2012

AID in Potsdam Standard

AID in Potsdam Standard



Introduction


AID in Potsdam Standard
UP12 aid in Potsdam Standard.png
BioBrick Nr. BBa_K929302
RFC standard [http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard RFC 10], [http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard Potsdam Standard]
Requirement pSB1C3
Source existing part:(BBa_K103001)
Submitted by [http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]


AID is known to be responsible for somatic hypermutation and the class-switch recombination of immunoglobulin in B cells. This enzyme of 28 kDa originally occurs in B cells but does also show activity after transfection into CHO cells. AID induces the deamination of cytidine to uridine at actively transcribed single strand DNA. The replacement of cytidine by uridine leads to a mismatch during DNA replication and integrates a single base substitution predominantly in the immunoglobulin genes.











Cloning with Potsdam Standard


To generate this part, we used the [http://2012.igem.org/Team:Potsdam_Bioware/Project/Potsdam_Standard Potsdam Standard] for cloning the AID into the Potsdam Standard Backbone. Here, we tested four different ligation conditions: 1. with electrophoretic unpurified, digested Potsdam Standard backbone with and without ligase and 2. with electrophoretic purified digested Potsdam Standard backbone with and without ligase. To compute the efficiency, we counted the colonies without and with red fluorescence indicating a failed ligation reaction (fig. 1). The results are summarized in fig. 2. We can see that with ligase the ligation success is much higher than without, and that a electrophoretic purification of the backbone improve the ligation efficiency.

Fig. 1: Result of the transformation in E. coli
Fig. 2: Relative ligation success of the four conditions







Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 84
    Illegal SapI site found at 185