Part:BBa_K3308010

[Tvo VMA C48 C]-[NrdJ-1 C (5-41)]-SEIVL-gpD

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]

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 C2 construct. The C-Terminus of this split intein, TvoVMA, is fused to a split site we have chosen in the C-terminus of the intein NrdJ-1.

Design

This construct has the C terminus of TvoVMA Intein, it is covalently attached to the second half of the C-terminal NrdJ-1 InteinBBa_K3308073. The main purpose of this construct is to preserve functional splicing of TvoVma N intein.

For TvoVMA we thought that it could better handle a C+2 change better than a N-1 change; therefore the TvoVMA was chosen to be put in this specific split site because the N and C flanking sequences on either side of the spliting site of NrdJ-1 C match what we expect is needed for efficient splicing.[3].

This construct was predicted to splice in the presence of its partner, BBa_K3308009, to form fully fucntional C-terminus NrdJ-1 (BBa_K3308012). If splicing occurs as planned between these two constructs then, addition of BBa_K3308009, BBa_K3308010, and BBa_K3308011 should result in the effective reconstruction of the full extein (GB1-GTNPC-SEIVL-gpD)- BBa_K3308013

Usage

Each construct of the set was labeled with 6XHis tag, for the purposes of purification via Ni-NTA resin(1ul/mL of culture). Following the His-tag the composite part also consists of a Tev7 Protease binding site, indicated the three dashed lines. It is important to note that the addition of the tag and cleavage site was not expected to have any impact on the splicing mechanisms of the intein.

This construct was induced and expected to react with BBa_K3308009 C2 to form the spliced product, the full terminus of the N- NrdJ-1 Intein CSP BBa_K3308012.

Results

Early on the expression and attempted purifications of this contruct, we noticed that levels were extremely low. There were low amount fo expression in comparison to constructs in the same nested inteins set. We found that many other intein researchers have found that the C- side of intein N ans C terminals are much more disordered [6].

Figure 2: Purification of K3308009( 30) and K3308010 (31) The above gel is a SDS-PAGE of the steps of purification. P- Pellet, S- Supernatant, Ft- Flowthrough, W-Wash(in sequential order), E- Elution] construct 31 corresponds with this composite part. There is some level of protein the elution; however, we cannot deduce the actual cocnentration of our protein of interest

The elution has very minimal to no protein in two type of lysis. Almsot all of the protein of interest was lost in the Pellet lane indicated in Figure 2 and 3. We concluded that this might mean that the disordered structure of C terminus of inteins (gp41-1-C) [6]. In presence of lysing and purifiation reagents, this C2 construct likely formed inclusion bodies(Insouble-therefore would show in the pellet).

Inorder to test whether lysis was the main issue contributing to why there was such little protein, we conducted a comparison between Emulsiflex lysis method versus our standard sonciation protocol. Figure 3, you can see although we optimized lysis, all of the protein remained in the pellet, indicating that formation of inclusion bodies was the main issue.

In response to C-terminal Intein insolubility, we decide to replicate these constructs into pTEV6/pKLD66 plasmid backbone BBa_K3308093 containing Maltose binding protein. Addition of maltose binding protein is found to increase the solubility of C-terminal inteins. This part is exactly the same as partinfo>BBa_K3308084</partinfo> ; however that MBP has been added to the N-terminus of the C2 Construct.

Figure 3: Purification of K3308010( 31) and K3308012 (33) The SDS-PAGE of the steps of purification. P- Pellet, S- Supernatant, Ft- Flowthrough, W-Wash(in sequential order), E- Elution] Almost all of the expressed protein, showing as the dark top band in the pellet, came of the pellet meaning that we were unable to solubilize the protein.

Figure 4: Purification via Emulsiflex of K3308010( 31) and K3308012 (33) this Figure is identical in lane composition as figure 2; P- Pellet, S- Supernatant, Ft- Flowthrough, W-Wash(in sequential order), E- Elution]. Even though lysis protocol were optimized, all the of protein came off in the Pellet.

The results from Figure 2 and 3, were a major component to the evolution of our contructs. The insolubility of the C-terminals drastically impacted whether or not we were able to isolate the protein in buffer. We find that there is good expression of the C terminal inteins in our nested system; the best way to visualize their presence and activity with its corresponding N terminal had to be in Lysate mixture. Consequentially, there was a lot of noise in the gels.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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

[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

[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

[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

[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] Shah, N. H., Eryilmaz, E., Cowburn, D., & Muir, T. W. (2013). Naturally split inteins assemble through a “capture and collapse” mechanism. Journal of the American Chemical Society, 135(49), 18673–18681. https://doi.org/10.1021/ja4104364

[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

This page was was last updated by Pittsburgh 2019 team.

This part is this set of nested Inteins constructs: BBa_K3308007. BBa_K3308008. BBa_K3308009. BBa_K3308011. BBa_K3308012. BBa_K3308013.