Difference between revisions of "Part:BBa K929003"

Latest revision as of 22:42, 29 September 2012

modified AID with CMV, hGH-polyA and eGFP

General information

CMV+mod. AID+eGFP+pA
BioBrick Nr.	BBa_K929003
RFC standard	RFC 10 with aditional AgeI restriciton site
Requirement	pSB1C3
Source	existing parts:(BBa_K929004; BBa_I712004; BBa_K404108)
Submitted by	[http://2012.igem.org/Team:Potsdam_Bioware Potsdam_Bioware2012]

Fig. 1 modified AID with CMV, hGH-polyA and eGFP in psB1C3 vector

The BioBrick "modified AID with CMV, hGH-polyA and eGFP" is an extended version of the existing BioBrick "modified AID+eGFP"(BBa_K929004). It is built of 3 parts: CMV promoter (BBa_I712004), modified AID and eGFP (BBa_K929004) and hGH polyadenylation signal sequence (BBa_K404108). It was designed for strong expression of the fusion protein modified AID+eGFP. In comparison to the part "modified AID with CMV and hGH polyA"(BBa_K929004) the green fluorescent reporter eGFP allows 1.)to check the transfection success 2.)sort transfected cells via FACS and 3.)to check the cellular localization of the fusion protein.

Related parts:
For using different promoters or terminators see BBa_K929004 (modified AID+eGFP), BBa_K929001 (just modified AID), its expression part BBa_K929001 (modified AID, CMV and hGH-polyadenylation sequence)or the expression part for wildtype AID BBa_K929000 (wtAID, CMV and hGH-polyadenylation sequence).

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.

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.

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

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)).

Green fluorescence reporter:
The modified AID was fused with eGFP to check 1.)the tranfection success 2.)the cellular lokalization of the improved AID and 3.) to select transfected cells via FACS.

• Transfection success and cellular localization:

Fig. 3: CHO cells were transfected A)with "modified AID+eGFP" (BBa_K929003)and B)with "modified AID+eGFP" (BBa_K929003) and a mCherry labeled nanobody construct (BBa_K929107)

The fluorescence microscope images(Fig. 3) show transfected cells A)with "modified AID+eGFP" (BBa_K929003)and B)with "modified AID+eGFP" (BBa_K929003) and a mCherry labeled nanobody construct (BBa_K929107)]]. "Modified AID+eGFP" (BBa_K929003)proved to be located in the nucleus. This shows that the deletion of the NES(Nuclear Export Sequence) and the addition of an NLS(Nuclear Localization Sequence) leads to an accumulation in the nucleus as it was planed. Therefore we would expect an increased mutation rate with "modified AID+eGFP" but as described above the experimental setup possible can not reflect this. It is also shown that transfected cells(green nucleus, Fig. 3 A&B) can be discriminated from not transfected (grey, Fig. 3). This can be used for a fast selection of transfected cells via FACS. Fig. 3 B shows that "modified AID+eGFP" can be used in combination with an other fluorescence labeld construct (mCherry labeled nanobody construct ,BBa_K929107) and co transfected cells can be easily found or selected via FACS.

Usage and Biology

Sequence and Features