Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Potsdam Bioware 2012 iGEM
Date created: 2012-09-17 11:00:00
Date modified: 2015-05-08 01:13:46
modified AID with CMV, hGH-polyA and eGFP
| Types | DnaRegion |
| Roles | Composite engineered_region |
| Sequences | BBa_K929003_sequence (Version 1) |
Description
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.
Notes
[[Image:UP12_biobrick3.jpg]]Source
4 parts: CMV promoter (BBa_I712004), superAID(BBa_K929001), eGFP (BBa_K404316) and hGH polyadenylation signal sequence (BBa_K404108)| Sequence Annotation | Location | Component / Role(s) |
| BBa_I712004 Kozak sequence BBa_K929001 BBa_K929004 AUG NLS sequence part of BBa_K103001 BBa_K404316 AgeI UAA hGH terminator BBa_K404108 | 1,654 663,677 663,1243 663,1966 671,673 678,696 697,1243 1244,1966 1958,1963 1964,1966 1975,2453 1975,2453 | feature/BioBrick engineered_region ribosome_entry_site feature/rbs engineered_region feature/BioBrick feature/BioBrick engineered_region feature/start start_codon sequence_feature feature/dna engineered_region feature/BioBrick feature/BioBrick engineered_region sequence_feature feature/dna stop_codon feature/stop polyA_site feature/polya feature/BioBrick engineered_region |