Part:BBa_K323061:Experience

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University of Minnesota 2012 Part characterization

• Registry part BBa_K323061 was chosen for characterization. It was submitted by the 2010 team from Slovenia as a reporter fusion between YFP and CFP, flanked by a promoter and terminator sequence.

• We initially intended to utilize this reporter fusion; however, after characterizing this part through multiple sequence alignments, we have concluded that the part submitted is not in fact a YFP::CFP fusion. Both of the open-reading frames (ORF) identified in this part are clearly YFP or GFP variants (Figure 1), displaying high similarity to established eGFP and eYFP ORFs used for this comparison. A few point mutations are observed at the nucleotide level; however, all of these produce silent mutations and no change in the ORF itself (Figure 1). All alignments were conducted using [http://multalin.toulouse.inra.fr/multalin/ MultAlin].

Figure 1: Multiple alignment of a presumed YFP::CFP fusion to eGFP and YFP. Part BBa_323061 was separated into two, separate ORFs for ease of comparison (named 1st ORF and 2nd ORF in the alignments). Perfect matches between the two aligned sequences are shown in red, while differences are shown in blue. A consensus from each alignment is shown on the bottom line.

• This high level of conservation was entirely different than the results obtained when the BBa_323061 part was compared to a CFP ORF. In that case, no significant similarity shared was observed (Figure 2). In both cases, many significant changes were observed throughout the ORF which lead to drastic changes in the translational product itself (data not shown).

Figure 2: Multiple alignment of a presumed YFP::CFP fusion to CFP. Part BBa_323061 was separated into two, separate ORFs for ease of comparison (named 1st ORF and 2nd ORF in the alignments). Perfect matches between the two aligned sequences are shown in red, while differences are shown in blue. A consensus from each alignment is shown on the bottom line.

Conclusions

• Based on this characterization, we have concluded that the submitted sequence for BBa_323061 is incorrect; it is not a YFP::CFP fusion. It is unclear at this point whether the sequence corresponds to a GFP or YFP ORF, but it is clear that this fusion is between some combination of those two fluorophors and not CFP.

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