Difference between revisions of "Part:BBa K929302"

Revision as of 13:49, 28 September 2012

AID in Potsdam Standard

AID in Potsdam Standard

Introduction

Potsdam Standard Cloning Vector
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