Part:BBa_K3308115

Lumio-AEY-[Npu-PCC73102 DnaE N (1-28)]-[Tvo VMA C48 N]

Lumio-AEY-[Npu-PCC73102 DnaE N (1-28)]-[Tvo VMA C48 N]

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 construct has the first half of the N-terminal NPU dnaE Intein, which means that it is covalently attached to the N-terminal extein,Lumino. In between the N-terminus and the extein we have inserted has consensus flanking sequences, AEY, that are published as essential to aiding splicing of NPU dnaE, once the whole terminal comes together forming.[6]. This part is meant to react with BBa_K3308116 . The main purpose of this construct is to use the use of NPU DnaE as our outer intein in our nested inteins project. We theorized that the robustness of Npu DnaE come from its block sites and ability to fold properly to facilitate the reaction under different conditions. We wanted to create a nested intein with it as the outside intein for this reason, to try and retain functionality in the second half of our nested inteins experiment.

The native junction sequence of TVO Vma has been changed from GK(N-2,N-1) to EC(N-2, N-1). Junction sequences are essential for inteins to splice. Non-native sequences can have adverse effects on the splicing mechanism. While we did not believe this new junction sequence was a good fit, we prioritized keeping NPU DnaE as our outer intein

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_K3308116 N2 to form the spliced product, the full terminus of the N- NPU dnaE Intein BBa_K3308119.

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.

Contribution Markup

This page was was last updated by Pittsburgh 2019 team.