Difference between revisions of "Part:BBa K3308027"

Revision as of 00:33, 22 October 2019

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

A-B Linker part

Overview

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 inducing the splicing o SceVMA to in turn form a fucntional extein(POI). Three orthogonal split-inteins A(blue), B (pink, C (green) are configured to splice a linker in series to form this linker

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. [4,6] .[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

Usage

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] Â 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), 11005â€“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.

This part is this set of nested Inteins constructs:

BBa_K3308028. BBa_K3308029. BBa_K3308030. BBa_K3308027. BBa_K3308032. BBa_K3308033. BBa_K3308034. BBa_K3308035. BBa_K3308036.

@@ Line 5: / Line 5: @@
 ===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.]]
+[[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 inducing the splicing o SceVMA to in turn form a fucntional extein(POI). Three orthogonal split-inteins A(blue), B (pink, C (green) are configured to splice a linker in series to form this linker]]
 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 [[#References|[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.