Difference between revisions of "Part:BBa K3308033"

Latest revision as of 03:42, 22 October 2019

split linker constructs:C-VMA-(gp41-1)

C-VMA-(gp41-1)

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]. 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 this construct splices with BBa_K3308028 and BBa_K3308032 to form the GS Linker.

Results

Figure 2: Purification of 44(this construct). This construct has N-terminal MBP; thus, we expected to see better solubility in purifications, this gel is purification of 200ml cultures indcued with IPTG for 4 hours at 37 degrees. This constuct was soluble in elution and presented in enough concentration in the elution to conduct diafiltration to filter out all other impurities before use in assays

This construct showed moderate evels of expression in pTEV6 backbone containing MBP. Once it was diafiltrated into splicing buffer, it was combined with part 64 (BBa_K3308034) which contains the C-terminal gp41-8 intein at the N teminal end and SceVMA-C and our extein GB1. In Figure 3, both gp41-8 terminal were combined and we do not a band formation of a band after the addition of the two construct at t= 18 hours. This was a negative result.

Figure 3:Splicing reaction between this 60(BBa_K3308033) and 64 (BBa_K3308034)In this SDS_PAGE we wanted to see if the expected splicing of gp41-8 then consequentially SceVMA to make the full extein MBP and GB1 was effected by temperature

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_K3308027. BBa_K3308028. BBa_K3308030. BBa_K3308029. BBa_K3308032. BBa_K3308031. BBa_K3308033. BBa_K3308034. BBa_K3308035. BBa_K3308036.