Difference between revisions of "Part:BBa K929002"

Latest revision as of 22:42, 29 September 2012

modified AID with CMV and hGH-polyA

General information

CMV modified AID pA
BioBrick Nr.	BBa_K929002
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]

Fig. 1: modified AID with CMV and hGH-polyA in pSB1C3 vector

The BioBrick "modified_AID with CMV and hGH-polyA" (BBa_K929002) 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.

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.You can fuse this part with RFC 25 parts(C-terminal of modified AID) but than you have to add hGH polyadenylation signal sequece again via serial cloning.To generate fusions (C-terminal of the modified AID)without CMV-promoter use BBa_K929001 or the existing fusion of modified AID with eGFP(BBa_K929004) that is also available with CMV-promoter and hGH polyadenlation sequence(BBa_K929003).

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.

Characterization

Mutation rate in CHO-cells:

Fig. 2: Comparison of the mutation rates between the wildtype AID, modified AID and modified AID-eGFP

We checked the mutation rate of wildtype AID(BBa_K929000), modified AID(BBa_K929002) and modified AID+eGFP(BBa_K929003) (all expressed with CMV-promoter and hGHpolyA). Therefor we cotransfected CHO cells with a single chain construct and one of the AID versions. After certain expression time we purified the the single chain plasmids and transformed them into E.coli to enrich the mutated plasmids. After overnight culture and purification of the transformed plasmids, samples where sequenced.
There is a significant higher mutation rate when wildtype AID, modified AID or modified AID-eGFP is added, compared to the same experiment without AID. Surprisingly, the mutation rate of wt AID is two times higher than the mutation rate of the modified AID or modified AID-eGFP (Fig. 2). This observation is contrary to our expectation. The modified AID+eGFP is located in the nucleus (Fig. 3), that means our construct works and should have a higher mutation rate. One possible explanation is that the modified AID has a very high mutation rate and therefore the transfected cells die or inactivate the plasmid like it was observed for the wildtype AID (Martin and Scharff (2002)).

Sequence and Features