Part:BBa_K929002

modified AID with CMV and hGH-polyA

CMV_mod. AID_pA
BioBrick Nr.	BBa_929002
RFC standard	RFC 10
Requirement	pSB1C3
Source	existing parts:(BBa_I712004; BBa_K929001; BBa_K404108)
Submitted by	[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]

The BioBrick "modified_AID with CMV and hGH-polyA" is an extended version of the existing BioBrick modified AID (BBa_K929001). It is built of 3 parts: CMV promoter (BBa_I712004), modified_AID (BBa_K929001) and hGH polyadenylation signal sequence (BBa_K404108).

AID:

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.

The AID motif is naturally terminated with the Nuclear Export Sequence (NES) that causes the protein to translocate from the nucleus to the cytoplasm. Additionally, upstream, dysfunctional Nuclear Localization Sequence (NLS) is located. Due to the fact that AID mutates the actively transcribed single stranded DNA, it is supposed that the direction of the enzyme to the inside of the nucleus would improve the mutation rate. That is why we called it superAID

Functional NLS sequence:

This part of the BioBrick directs the expressed protein into the nucleus, where it can mutate stronger.

Kozak sequence

Kozak consensus sequence is added upstream of the AID mutant to express the protein stronger.

Aditional AgeI restriciton site

An aditional AgeI restriciton site was added at the 3` end in front of the stop codon "taa" of the modified AID. Because a hGH polyadenylation signal sequece was added via serial cloning, this restiriction site is no longer usefull. To generate fusions (C-terminal of the modified AID) use BBa_K or the existing fusion of modified AID with eGFP(BBa_929004) that is also available with CMV-promoter and hGH polyadenlation sequence(BBa_929004)

CMV promoter:

CMV is an immediate-early Cytomegalovirus promoter for high-level expression. The CMV promoter is commonly used due to its very strong activity and effectivity in a broad range of cell types. The BioBrick is therefore improved via addition of the strong promoter.

hGH polyadenylation signal sequence:

Polyadenylation is a significant part for the translation and stability of mRNA. In eukaryotes, it is part of the process that produces mature messenger RNA (mRNA) for translation. It, therefore, forms part of the larger process of gene expression. hGH terminator gives a signal to start polyadenylation in the translation process.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1241
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1643
  Illegal BsaI.rc site found at 770
  Illegal SapI site found at 871