Part:BBa_K3308027

Split linker constructs:N-VMA-(gp41-1)

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]

File:Split linker- general concept.png

Figure 1: Nesting NrdJ-1 Inteins with gp41-1 and TvoVMA split inteins.This composite part contains the N-terminal of primary splicing intein, TvoVMA, on the C side. We have denoted it as the C1 construct. The N-Terminus of this split intein is fused to a split site we have chosen in the C-terminus of the intein NrdJ-1. In order to choose this split site we had to consider that the N-terminal flanking sequences for TvoVMA inteins.

Results

Figure 3: On this gel we tested diafiltration of pVS30(BBa_K3308010) in the last two lanes The above gel is a SDS-PAGE of the last step of purification E- Elution comapred to after the elution was dialyzed

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
  Illegal XhoI site found at 1975
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 106
  Illegal SapI.rc site found at 1944
  

Split linker constructs: AC: gp41-1-GS linker-gp41-8

Overview

[[File:split-linker general concept 44-68.png|920px|thumb|center|Figure 1: Reconstitution of exteins with linker dependent SceVMA splicing. We utilize high affinity controllable intein splicing to reconstitute a GS Linker bring together weakly associating proximity induced inteins SceVMA. Formation of this GS Linker will bring SceVMA terminals in close proximity to each other, increasing the effective concentration.]

The Pittsburgh iGEM team 2019 designed two approaches to creating a intein based circuit system. The second system, we have name "split-linker", was inspired after we began designing nested intein cosntructs. We found that it was relatively difficult to identify good location to split an extein. The stie at which the extein was split had to match a proposed flanking sequence necessary for the splicing of inteins adjacent to that extein [3].We find that there is a necessary comprimise between maintaining the extein sequence and maintaning the intein's flanking sequence. This system was designed to preserve the native flanking sequences of the exteins.

Design

Our work is largely inspired by literature on the "proximity induced" Sce VMA split intein. Here are two papers highlighting the applications of this intein: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.[2,4,6]

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

Results

Assembly Compatibility:

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

  Illegal BglII site found at 408
  Illegal BglII site found at 1198
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 106
  

References

[1] Shah, N. H., Dann, G. P., Vila-Perelló, M., Liu, Z., & Muir, T. W. (2012). Ultrafast protein splicing is common among cyanobacterial split inteins: Implications for protein engineering. Journal of the American Chemical Society, 134(28), 11338–11341. https://doi.org/10.1021/ja303226x

[2] Mootz, H. D., & Muir, T. W. (2002). Protein splicing triggered by a small molecule. Journal of the American Chemical Society, 124(31), 9044-5. https://doi.org/10.1021/ja026769o

[3]  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

[4] Selgrade, D. F., Lohmueller, J. J., Lienert, F., & Silver, P. A. (2013). Protein scaffold-activated protein trans-splicing in mammalian cells. Journal of the American Chemical Society, 135(20), 7713-7719. https://doi.org/10.1021/ja401689b

[6] Tyszkiewicz, A. B., & Muir, T. W. (2008). Activation of protein splicing with light in yeast. Nature Methods, 5(4), 303-305. https://doi.org/10.1038/nmeth.1189

[7] Gramespacher, J. A., Stevens, A. J., Nguyen, D. P., Chin, J. W., & Muir, T. W. (2017). Intein Zymogens: Conditional Assembly and Splicing of Split Inteins via Targeted Proteolysis. Journal of the American Chemical Society, 139(24), 8074-8077. https://doi.org/10.1021/jacs.7b02618

Contribution Markup

This page was was last updated by Pittsburgh 2019 team.

Contribution Markup

This page was was last updated by Pittsburgh 2019 team.

This part is this set of nested Inteins constructs:

BBa_K3308028. No part name specified with partinfo tag.. No part name specified with partinfo tag.. BBa_K3308027. BBa_K3308032. No part name specified with partinfo tag.. No part name specified with partinfo tag.. No part name specified with partinfo tag.. No part name specified with partinfo tag..