Part:BBa_K3308118

Overview

The Pittsburgh iGEM team 2019 designed a modular protein circuit system consisting of split Intein-based logic gates. This composite part is an input of the proposed nested intein system. This system is composed of two-independent splicing events reconstituting function functional half of a nested intein. Each nested intein’s chain (N and C terminus) will be split at one location by another split intein rendering it nonfunctional. Consequently only splicing of the “inner inteins”, will reconstruct the functional intein that is fused to the desired extein. [5]In this system, the primary splicing events taking place at each split site of the nested intein halves, will serve an AND gate. Each AND is composed of two inputs, the N- and C- terminals of matching inteins.[1]

Design

This part was the second half of the C-terminal of the npu DnaE intein, which means that it is covalently attached to the C-terminal extein, Lumio. In between the C-terminus and the extein we have inserted the native flanking sequence, CFN, which are essential to aiding in the splicing once the whole terminal comes together[6]. The main purpose of this construct was to construct a functioning C terminal for Npu dnaE N-terminal BBa_K3308115 and BBa_K3308116. Unfortunately, no appropriate split sites with a C+1 native junction sequence to be found, to get around this we introduced a point mutation. A Blosum point mutation was used to create an artificial C+1 junction.Our main concerned with doing a mutation was affecting DnaE’s ability to fold properly to carry out its splicing mechanism. To prevent this, the mutation was in a region that avoided block sites which are known sites for facilitating the reaction mechanism as well as in a place where we believed the structure context would not affect the folding of Npu DnaE. With this new amino acid the first step of the splicing mechanism could now take place. The point mutation allowed for a matching C+2 junction site and a good spot according to our structure theory. We were, however, concerned with the N-1 side, but with little choices in terms of splice sites and already having made one point mutation we believed it was best to leave this junction unchanged.

Usage

Each construct of the set was labeled with 6XHis tag, for the purposes of purification via Ni-NTA resin(1ul/mL of culture). Following the His-tag the composite part also consists of a Tev7 Protease binding site, indicated the three dashed lines. It is important to note that the addition of the tag and cleavage site was not expected to have any impact on the splicing mechanisms of the intein. This construct was induced and expected to react with BBa_K3308117 C1 to form the spliced product, the full terminus of the C- NPU dnaE Intein BBa_K3308120 that has a point mutation from Valine to Serine on the 20th amino acid.

Results

Unfortunately, this part was unable to be Gibson Cloned correctly.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 408
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 106
  

References

[1] Gramespacher, J. A., Stevens, A. J., Thompson, R. E., & Muir, T. W. (2018). Improved protein splicing using embedded split inteins. Protein Science, 27(3), 614–619. https://doi.org/10.1002/pro.3357

[2] Beyer, H.M., Mikula, K.M., Li, M.,Wlodawer, A., Iwai, H., (2019) The crystal structure of the naturally split gp41-1 intein guides the engineering of orthogonal split inteins from a cis-splicing intein.BioRxiv. https://doi.org/10.1101/546465

[3] Lockless, S. W., & Muir, T. W. (2009). Traceless protein splicing utilizing evolved split inteins. Proceedings of the National Academy of Sciences of the United States of America, 106(27), 10999–11004. https://doi.org/10.1073/pnas.0902964106

[4] Amitai, G., Callahan, B. P., Stanger, M. J., Belfort, G., & Belfort, M. (2009). Modulation of intein activity by its neighboring extein substrates. Proceedings of the National Academy of Sciences, 106(27), 11005–11010. https://doi.org/10.1073/pnas.0904366106

[5] Appleby-Tagoe, J. H., Thiel, I. V., Wang, Y., Wang, Y., Mootz, H. D., & Liu, X. Q. (2011). Highly efficient and more general cis- and trans-splicing inteins through sequential directed evolution. Journal of Biological Chemistry, 286(39), 34440–34447. https://doi.org/10.1074/jbc.M111.277350

[6] Perler, F. B. (2002). InBase, the Intein Database. Nucleic Acids Res. 30, 383-384.