Part:BBa_K3308013

GB1-GTNPC-SEIVL-gpD

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 the N-terminal of primary splicing intein, gp41-1. We have denoted it as the MSP construct. This costruct is the positive control of functional splciign fo NrdJ-1. If there is SPlicing between NSP and CSP then the Mixed spliced product (this part) would be constructed

Design

This composite part contains the Full extein sequence, The N and C terminal Exteins, GB1 and gpD

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, SEIVL-gpD, the same as BBa_K3308010.[2,3,4]

This part is the expected product of functional splicing of TvoVMA and gp41-1. inteins which will brign together functional splicing of external NrdJ-1 intein.

Usage

The main purpose of this part was to act as a positive control for splicing of composite parts BBa_K3308011 and BBa_K3308012

In the test of the full nested intein system when N1, N2, C1, and C2 are all added together sequentially NSP and CSP should be made, then those splicign will in turn produce this composite part MSP N1BBa_K3308007

N2BBa_K3308008

NSP BBa_K3308011

C1BBa_K3308009

C2BBa_K3308010

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

This composite part is only consiting of the exteins and the corresponding N and C flanking sequences necessary for NrdJ-1. There were no issues with purification of this part.

Figure 3: Purification via Sonification of K3308009( 30) and K3308013 (34) P- Pellet, S- Supernatant, Ft- Flowthrough, W-Wash, E- Elution. We were able to efficiently purify this construct to use as control for the detecting splicing of the nested NrdJ-1 system

The results from Figure 2 and 3, were a major component to the evolution of our contructs. The insolubility of the C-terminals drastically impacted whether or not we were able to isolate the protein in buffer. We find that there is good expression of the C terminal inteins in our nested system; the best way to visualize their presence and activity with its corresponding N terminal had to be in Lysate mixture. Consequentially, there was a lot of noise in the gels.

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_K3308011. BBa_K3308012.