Part:BBa_K3308008

[gp41-1 C]-[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 1: Nesting NrdJ-1 Inteins with gp41-1 and TvoVMA split inteins.This composite part contains the C-terminal of primary splicing intein, gp41-1. We have denoted it as the N2 construct. The C-Terminus of this split intein is fused to a split site we have chosen in the N-terminus of the intein NrdJ-1. In order to choose this split site we had to consider that the C-terminal flanking sequences for gp41-1 inteins, as well as the predicted structure of the NrdJ-1 intein.

Design

This construct has C terminal of gp41-1 Intein, which means that it is covalently attached to the second half of the N-terminal NrdJ-1 InteinBBa_K3308069. This sepcific split site was chosen in N-NrdJ-1 because it provided the C1 requisite C or S amino acid.[2,3,4] The main purpose of this construct is to preserve functional splicing of gp41-1 C intein.[1]

When looking into the block B site of gp41-1, we saw that there were many acid and base amino acid since this block is involved in the first step as reviewed above we thought that it would be more important than the N-1 intein here. [2] Considering these things and the fact that any percent yield would still be considered a success for our current methods we decided that this site would be a strong candidate for success.

This construct was predicted to splice in the presence of its partner, BBa_K3308007, to form fully fucntional N-terminus NrdJ-1 (BBa_K3308011). If splicing occurs as planned betweenn these two constructs then, addition of BBa_K3308007, BBa_K3308008, and BBa_K3308012 should result in the effective reconstruction of the full extein (GB1-GTNPC-SEIVL-gpD)- BBa_K3308013

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_K3308007 N2 to form the spliced product, the full terminus of the N- NrdJ-1 Intein NSP ( BBa_K3308011.

Results

Figure 2: Purification of K3308008 The above gel is a SDS-PAGE of the steps of purification. P- Pellet, S- Supernatant, Ft- Flowthrough, W-Wash(in sequential order), E- Elution

Early on the expression and attempted purifications of this contruct, we noticed that levels were low. There were low amount fo expression in comparison to constructs in the same nested inteins set. We found that many other intein researchers have found that the C- side of intein N ans C terminals are much more disordered [6]

The elution has very minimal to no protein. Almsot all of the protein of interest was lost in the Pellet lane indicated in Figure 2. We concluded that this might mean that the disordered structure of C terminus of inteins (gp41-1-C) [6]. All other C-terminal full inteins also showed difficulty in purifications BBa_K3308011, BBa_K3308010, BBa_K3308012. In presence of lysing and purifiation reagents, this N2 construct liekly formed inclusion bodies(Insouble-therefore would show in the pellet).

We also tried to utilize BugBuster Lysis reagent made by ThermoFisher, which claimed to have increased ability to solubility of proteins that form inclusion bodies. We found that both methods were effecient in lysis of cells; however, we were unable to conclude whether solubility was changed in anyway.

In response to C-terminal Intein insolubility, we decide to replicate these constructs into pTEV6/pKLD66 plasmid backbone BBa_K3308093 containing Maltose binding protein. Addition of maltose binding protein is found to increase the solubility of C-terminal inteins. This part is exactly the same as BBa_K3308082; however that MBP has been added to the N-terminus of the N2 Construct.

Sequence and Features

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., Eryilmaz, E., Cowburn, D., & Muir, T. W. (2013). Naturally split inteins assemble through a “capture and collapse” mechanism. Journal of the American Chemical Society, 135(49), 18673–18681. https://doi.org/10.1021/ja4104364

[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_K3308009. BBa_K3308010. BBa_K3308011. BBa_K3308012. BBa_K3308013.