Part:BBa_K3308130
[Npu-PCC73102 DnaE C (20-36)]
[Npu-PCC73102 DnaE C (20-36)]
Overview
During the design of this construct, the 2019 Pittsburgh iGEM Team had trouble finding an appropriate split site for Npu DNAE C terminal half. In order to find an appropriate split site, we needed to preserve the C+1 amino acid in the flanking sequence for the intein, since this amino acid residue is required for successful splicing of the intein. In order to circumvent this, we introduced a mutation into the sequence of Npu DNAE in order to include the essential C+1 amino acid residue for successful splicing of NrdJ-1.
Design
Using the Blosum Scoring Matrix[1], we analyzed which amino acid residue change would result in the least disruption to the intein secondary and tertiary structure. The Blosum Score Matrix yielded a -2 score for a V-->S mutation. Although this score was not ideal, after analyzing the structure context, we concluded that this substitution would not severely affect refolding and functionality of the intein. We determined that the amino acid mutation would be a change from Valine to Serine in the Npu DNAE sequence at the C+1 junction site. The C+1 junction site is involved in the first step of the splicing mechanism and is important in order to allow for successful splicing to occur[2].
References
[1] Henikoff, S., & Henikoff, J. G. (1992). Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences of the United States of America, 89(22), 10915–10919. doi:10.1073/pnas.89.22.10915
[2] Oemig, Jesper S. (2013). Structural Studies on Inteins. (National Doctoral Programme in Informational and Structural Biology). University of Helsinki, Helsinki, Finland.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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