Composite

Part:BBa_K929003

Designed by: Potsdam Bioware 2012 iGEM   Group: iGEM12_Potsdam_Bioware   (2012-09-18)
Revision as of 12:59, 18 September 2012 by Registry (Talk | contribs)

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modified AID with CMV, hGH-polyA and eGFP

The BioBrick wtAID is an extended version of the existing AID BioBrick (BBa_K929001). It is built of 3 parts: CMV promoter (BBa_I712004), superAID, eGFP (BBa_K404316) 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.

CMV promoter:

CMV stands for cytomegalovirus. 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.

eGFP:

In order to test where and if the AID modified sequence is functional we added to it a eGFP protein. It is used as a marker gene for detection of transfected cells, e.g. tumor cells.


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 1958
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2360
    Illegal BsaI.rc site found at 770
    Illegal BsaI.rc site found at 1887
    Illegal SapI site found at 871


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Parameters
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