Difference between revisions of "Part:BBa K3308027"

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		+	__NOTOC__
		+	<partinfo>BBa_K3308031 short</partinfo>
		+	===C1-construct===
		+	===Overview===
		+	[[File:Coded- Nested intein diagram.png|300px|thumb|right| ]] 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. [[#References|[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.[[#References|[1]]]
		+
		+	[[File:split linker- general concept.png|945px|thumb|center|'''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.]]
		+
		+	===Design===
		+
		+
		+
		+
		+	===Usage===
		+
		+
		+	===Results===
		+
		+	[[File:pHG68.png|600px|thumb|right|'''Figure 3: On this gel we tested diafiltration of pVS30(<partinfo>BBa_K3308010</partinfo>) 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]]
		+
		+
		+
		+
		+
		+	<!-- -->
		+	<span class='h3bb'>Sequence and Features</span>
		+	<partinfo>BBa_K3308031 SequenceAndFeatures</partinfo>
		+
	__NOTOC__		__NOTOC__
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	===A-B Linker part===		===A-B Linker part===
	===Overview===		===Overview===
−	[[File:split-linker general concept 44-68.png|920px|thumb|center|'''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]]	+	[[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.]
−	[[#References|[1]]]	+	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.
−		+
−	[[File:pvs000028-34.png|900px|thumb|center|'''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]]	+
	===Design===		===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.[[#References|[2,4,6]]]
		+
		+	[[File:pvs000028-34.png|900px|thumb|center|'''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===		===Usage===
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	<!-- -->		<!-- -->
	<partinfo>BBa_K3308027 SequenceAndFeatures</partinfo>		<partinfo>BBa_K3308027 SequenceAndFeatures</partinfo>
		+
	===References===		===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	+	[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] 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	+	[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] 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	+	[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]  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
−	[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] 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
−	[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] 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
−	[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===		===Contribution Markup===
	This page was was last updated by Pittsburgh 2019 team.		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:
		+
		+	<partinfo>BBa_K3308028</partinfo>.
		+	<partinfo> BBa_K3308029 </partinfo>.
		+	<partinfo> BBa_K3308030 </partinfo>.
		+	<partinfo>BBa_K3308027</partinfo>.
		+	<partinfo>BBa_K3308032</partinfo>.
		+	<partinfo> BBa_K3308033 </partinfo>.
		+	<partinfo> BBa_K3308034 </partinfo>.
		+	<partinfo> BBa_K3308035 </partinfo>.
		+	<partinfo> BBa_K3308036 </partinfo>.

---

Revision as of 00:05, 22 October 2019

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

C1-construct

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]

Design

Usage

Results

Sequence and Features

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

A-B Linker part

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]

Usage

Results

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..