Part:BBa_K3308027

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

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.[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 gp41-1. 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 gp41-1 will splice with the N terminal gp41-1 in BBa_K3308031, and at the C teminal of this composite part consists of N-terminal gp41-8 intein and its corresponsng NF. The N terminal gp41-8 is to react with its C- gp41-8 in BBa_K3308034

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 was promising and showed good levels 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 werecombined and we see a band forming after the addition of the two construct at t= 18 hours. There is a clear depletion of starting constructs and we were also able to idenity the C- intein(C-gp4-1). The best splicing temperature is hard to conclude because the spliced product was made in all of them. This proves that a system like this can liekly be utilize in a variety of exteins that might be temperature dependent for proper folding. lane 44 and 64 are the negative controls of this reaction because the intein pairs have yet to be added together.

In Figure 4, we are testing the kinetic of this reaction based on the formation of the spliced product. Lane 0 are the negative controls of this reactions, and well the 4hr samples of the separate inteins. The spliced product shows in on the gradent SDS-PAGE get at the appropriate size of 64.84 kDa. These results can be concluded as postive results of the splicing of gp41-8 only with the presence of the flanking sequence. It is important to understant that Intein need many biochemcial variable to be correct in order to splice properly. Here we see that only with the requisite of native flanking sequence being preserved splicing occurred. The band shows up within 5 minutes of combining the constructs; however after band intensity analysis we were able to conclude that the splicing efficiency was only at 50 compared to reported 85-90% in papers discussing gp41-8.

Figure 3:Splicing reaction between this 44(BBa_K3308027) and 64 (BBa_K3308034)In this SDS_PAGE we wanted to see if the expected splicing of gp41-8 was effected by temperature

Figure 4: Kinetics: splicing reaction between this 44(BBa_K3308027) and 64 (BBa_K3308034)This SDS-PAGE shows that Intiein splicing of gp41-8 occured within 5 minutes of combining both termini in splicing buffer at 37° C

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_K3308029. BBa_K3308030. BBa_K3308027. BBa_K3308032. BBa_K3308033. BBa_K3308034. BBa_K3308035. BBa_K3308036.