Difference between revisions of "Part:BBa K929004"

Revision as of 14:01, 26 September 2012

modified AID+eGFP

General information

modified_AID+eGFP
BioBrick Nr.	BBa_K929004
RFC standard	RFC 10 and aditional AgeI site
Requirement	pSB1C3
Source	existing parts: BBa_K404316 and BBa_K929001
Submitted by	[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]

This is a composite part from the existing parts: modified_AID (BBa_K929001) and eGFP (BBa_K404316). Modified_AID (BBa_K929001) is in RFC 10 cloning standard and has an aditional AgeI restriciton site. Therefore it can be fused with RFC 25 parts (C-terminal of modified AID), see Part_Design. This part was not used to express the fusion protein. It is an usefull intermediate that can be combined with differnt promoters and terminators. We combined this part with CMV-promoter and polyA-hGH (poly adenylation signal sequence)--> see BBa_929002.

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.

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.

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.

Aditional AgeI restriciton site This part has an aditional AgeI restriction site because its precursor "modified AID+eGFP"(BBa_K929004) was build of an RFC 25 part that has an AgeI restriction site in front of its stop codon. Therefore incompatibility with RFC 25 is displayed. Actually fusion with RFC 25 parts is possible (C-terminal of CMV-modified AID-eGFP) but hGH polyadenylatiosequence must be added again. CMV promoter and hGH-polyadenylation sequence were added via serial cloning (see PartDesign).

Characterization

To characterize the modified AID+eGFP fusion protein, we added a (strong) CMV promoter and a hGH polyadenylation sequence. See the characterization of the resulting part "modified AID with CMV, hGH-polyA and eGFP" (BBa_K929003).

Sequence and Features