Part:BBa_K3308011

GB1-GTNPC-[NrdJ-1 N (1-4)]-[NrdJ-1 N (5-104)]

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]

Figure 2: Nesting NrdJ-1 Inteins with gp41-1 and TvoVMA split inteins.This composite part contains full N-terminal of NrdJ-1. We have denoted it as the NSP construct. This costruct acts a positive control of splicing of gp41-1

Design

This composite part contains the N-terminal of primary splicing intein, gp41-1. We have denoted it as the NSP construct. This costruct acts a positive control of splicing of gp41-1 (BBa_K3308007 and BBa_K3308008). The part is the full NrdJ-1 N intein containing the total 104 amino acids. The extein we have inserted still has a consensus flanking sequences, GTNPC, the same as BBa_K3308007.[2,3,4]

This part is the expected product of functional splicing of gp41-1 intein .[4, 5] We also constructe this part to be able to test the when one the intein terminals is split, it can be added in solution with the full compliment to get functional reconstitution of the extein sequences BBa_K3308012. This part also had the same design consideration for insertion of a flanking sequence between the extein the NrdJ-1 N intein in part BBa_K3308007 .[2]

Usage

The main purpose of this part was to act as apositive control for splicing of composite parts BBa_K3308007 and BBa_K3308008 and also a functional N-intein NrdJ-1 that should be able to splice with the spliced product of BBa_K3308009 and BBa_K3308010 ( aka. CSP-BBa_K3308012 )

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.

Results

Figure 2: Full NrdJ-1- N Intein Purification efficiency pvs000043 (K3308011) We were able to purify and isolate relatively low concentration at a good purify in the elution.

This part purified relatively well and was later used in testing that once a nested C terminal NrdJ-1 came together it can fucntioanlyl splice with this composite part to form the full extein sequence. In Figure2, we can clearly see that all fo the protein of interest was extracted out of the pellet. We concluded that this likely had to do with enhanced ability of Nickel resin to bind to the His tag on the N terminals of the proteins. This results also questioned our method of purification in that Histidine residues in the cosntruct itself could be interring with nickle binding.

In future experiments we hope to add the parts BBa_K3308010 and BBa_K3308011 or BBa_K3308083 and BBa_K3308084 In a one pot reaction with this composite part. We should yield the same MSP, spliced product BBa_K3308013.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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] Shah, N. H., & Muir, T. W. (2014). Inteins: Nature’s gift to protein chemists. Chemical Science, 5(2), 446–461. https://doi.org/10.1039/c3sc52951g

[7] Øemig, J. S. (2013)Structural Studies on Intein. (Published Doctoral Dissertation). University of Helsinki. Helsinki, Finland Retrieved from https://pdfs.semanticscholar.org/3c6a/b9fa31488316df5f421869163101ba13037e.pdf

Contribution Markup

This page was was last updated by Pittsburgh 2019 team.

This part is this set of nested Inteins constructs: BBa_K3308007. BBa_K3308008. BBa_K3308009. BBa_K3308010. BBa_K3308012. BBa_K3308013.