Difference between revisions of "Part:BBa K3308028"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3308028 short</partinfo>
 
<partinfo>BBa_K3308028 short</partinfo>
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===A-B Linker part===
  
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Split LInker constructs: AC: gp41-1-GS linker-NrdJ-1
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===Overview===
  
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<!-- Add more about the biology of this part here
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[[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]]
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===Usage and Biology===
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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 site 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.
 +
 
 +
===Design===
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Our work is largely inspired by literature on the "proximity induced" Sce VMA split intein.[[#References|[2,4,6]]]. In the design process of this system we had to use orthogonal inteins; we referenced a recent discovery of orthogonal fast intein to utilize in this system [[#References|[8]]]  . This construct contains the C terminal intein of gp41-8. and its flanking sequence bdenoted as CF. We assume that the inclusion of flanking sequeence is suffiencient is preserving splicing of the linker[[#References|[1]]] , and this was the main concept to prove because other different SceVMA linker system have data to support effective splicing following construction of the linker.
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===Usage===
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This part is inovled in three-lart ligation varient of this system. We expect that the C terminal gp41-8 will splice with the N terminal gp41-8 in <partinfo>BBa_K3308032</partinfo>, and at the C teminal of this composite part consists of N-terminal gp41-1 intein and its corresponsng NF. The N terminal gp41-1 is to react with its C- gp41-1  in <partinfo>BBa_K3308033</partinfo>
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===Results===
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Unfortunately we were restricted on time to clone this contruct.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K3308028 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3308028 SequenceAndFeatures</partinfo>
  
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===References===
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[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
  
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<!-- Uncomment this to enable Functional Parameter display
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[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
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===Functional Parameters===
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<partinfo>BBa_K3308028 parameters</partinfo>
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[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
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[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
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[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
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[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
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[8] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D., & Schmidt, S. R. (2012). Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. Journal of Biological Chemistry, 287(34), 28686-28696. https://doi.org/10.1074/jbc.M112.372680
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===Contribution Markup===
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This page was was last updated by Pittsburgh 2019 team.
 +
 
 +
This part is this set of nested Inteins constructs:
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 +
<partinfo>BBa_K3308028</partinfo>.
 +
<partinfo>BBa_K3308027</partinfo>.
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<partinfo>BBa_K3308030</partinfo>.
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<partinfo>BBa_K3308029</partinfo>.
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<partinfo>BBa_K3308032</partinfo>.
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<partinfo>BBa_K3308033</partinfo>.
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<partinfo>BBa_K3308034</partinfo>.
 +
<partinfo>BBa_K3308035</partinfo>.
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<partinfo>BBa_K3308036</partinfo>.

Latest revision as of 02:27, 22 October 2019


Split LInker constructs: AB: gp41-1-GS linker-NrdJ-1

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 site 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.[2,4,6]. In the design process of this system we had to use orthogonal inteins; we referenced a recent discovery of orthogonal fast intein to utilize in this system [8] . This construct contains the C terminal intein of gp41-8. and its flanking sequence bdenoted as CF. We assume that the inclusion of flanking sequeence is suffiencient is preserving splicing of the linker[1] , and this was the main concept to prove because other different SceVMA linker system have data to support effective splicing following construction of the linker.

Usage

This part is inovled in three-lart ligation varient of this system. We expect that the C terminal gp41-8 will splice with the N terminal gp41-8 in BBa_K3308032, and at the C teminal of this composite part consists of N-terminal gp41-1 intein and its corresponsng NF. The N terminal gp41-1 is to react with its C- gp41-1 in BBa_K3308033

Results

Unfortunately we were restricted on time to clone this contruct.


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

[8] Carvajal-Vallejos, P., Pallissé, R., Mootz, H. D., & Schmidt, S. R. (2012). Unprecedented rates and efficiencies revealed for new natural split inteins from metagenomic sources. Journal of Biological Chemistry, 287(34), 28686-28696. https://doi.org/10.1074/jbc.M112.372680

Contribution Markup

This page was was last updated by Pittsburgh 2019 team.

This part is this set of nested Inteins constructs:

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