Part:BBa_K2005050
mCherry (oxidation-resistant)
mCherry red fluorescent protein (RFP) with its coding DNA sequence redesigned to minimize its susceptibility to oxidative DNA mutation. Compared with standard mCherry, this gene is therefore more stable and less likely to evolve out of a population of cells.
Sequence Design
To generate this artificial DNA sequence, we employed our custom-made algorithm for modifying gene sequences to eliminate mutagenic sites. These sites were identified based on prior research on DNA oxidation, which identified poly-guanine motifs as particularly vulnerable. At these motifs, synonymous codon substitutions were made to eliminate oxidation-prone nucleotide sequences and replace them with ones with a lower mutation risk. In addition, our algorithm used heuristics to factor in the effect different codons have on gene expression to ensure that the gene not only produced the same protein but produced it at similar levels. For this sequence, our algorithm could eliminate 65% of poly-guanine motifs and an overall reduction in oxidizable guanines.
Assembly
This sequence was synthesized and ligated into pSB1C3. We confirmed that this construct was synthesized and integrated correctly by sequencing. We cloned this gene with a T7 promoter (K2005061) to verify that both the function and expression of the fluorescent protein.
Mutagenicity
We quantified levels of oxidized guanine using mass spectrometry on purified samples of optimized mCherry. Comparison to the control mCherry Bba_J06504 verified that our optimization of this sequence reduced the rate of mutation. Please see our 2016 wiki for more details.
References
Senthilkumar, K., Grozema, F.C., Guerra, C.F., Bickelhaupt, F.M., and Siebbeles, L.D.A. (2003). Mapping the sites for selective oxidation of guanines in DNA. J. Am. Chem. Soc. 125, 13658–13659.
Improvement
Group: iGEM17_SUSTech_Shenzhen
Author: iGEM17_SUSTech_Shenzhen
Summary:We found that the part BBa_K2005050 is mCherry submitted by iGEM16_Vanderbilt. However, this mCherry is not designed for C.elegans. Thus, we got the sequence and improved it with C.elegans codon adaptor[1]. Compared with original mCherry, new synthesis mCherry sequence is modified in: a.adding three introns b.some codons modified The mCherry part we used now is optimized for C.elegans and perform excellently observed under confocal microscope. We added the figure to part page The whole neuron with dendrite can be observed distinctly. The branch of dendrite near the mouse can be observed as well(Figure). Universal mCherry coding sequence of three introns for capacity of expression in C.elegans. MCherry is a commonly used reporter. Since ordinary mCherry may not have high expression in C.elegans, artificial introns are inserted in the constructed plasmid[1]. We can obtain ord-10::Chrimson::GEM-GECO::mCherry transgenic offsprings microinjection. MCherry driven by promoter ord-10(Part:BBa_K2492004[1]) indicates the position of neuron AWA. Moreover the fluorescence of mCherry aids the imaging for the neuron AWA pattern. The intensity of fluorescence reveals the expression level approximately. [1]:S.Redemann. (2011). codon adaptation– based control of protein expression in C. elegans, Nature Methods, Vol.8 No.3 250-254.
More details about our test is in experience page[2]. And our improved new biobrick part is called BBa K2492002[3].
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