Composite
CSP

Part:BBa_K3308086

Designed by: Jemy Varghese, Harrison Green, Ripal Sheth, Victor So, Mel Marciesky   Group: iGEM19_Pittsburgh   (2019-10-06)
Revision as of 02:16, 21 October 2019 by Jvargh (Talk | contribs) (→‎Design)

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[NrdJ-1 C (1-4)]-[NrdJ-1 C (5-41)]-SEIVL-gpD

CSP-construct

Overview

Coded- Nested intein diagram.png
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 2: Nesting NrdJ-1 Inteins with gp41-1 and TvoVMA split inteins. This set of constructs is identical to BBa_K3308007-13; however,this this composite part contains a solubility tag (maltose binding protein) that is expected to aide in the solubilization of the parts inside the cell. The addition of this tag is said to decrease aggregation of proteins. [3]. This composite part contains the Full C-terminal of NrdJ-1. We have denoted it as the CSP construct. This costruct acts a positive control of splicing of TwoVMA.

Design

After expression and attempted purification of BBa_K3308081, We decided that we would conduct Gibson Assembly of the part into a different plasmid backbone BBa_K3308093 consisting of Maltose Binding Protein[3,5]. Maltose Bidning Protein is a relatively large 42 kDa. This composite part contains the N-terminal of primary splicing intein, gp41-1. We have denoted it as the NSP construct. This costruct acts a positive control of splicing of gp41-1 (BBa_K33080081 and BBa_K3308082). The part is the full NrdJ-1 N intein containing the total 104 amino acids. The extein we have inserted still has a consensus flanking sequences, SEIVL-gpD, the same as BBa_K3308084.[2,3,4] This part is the expected product of functional splicing of TvoVMA.[4, 5] We also constructe this part to be able to test the when one the intein terminals is split, it can be added in solution with the full compliment to get functional reconstitution of the extein sequences BBa_K3308086.

Usage

The main purpose of this part was to act as a positive control for splicing of composite parts BBa_K3308009 and BBa_K3308010 and also a functional N-intein NrdJ-1 that should be able to splice with the spliced product of BBa_K3308081 and BBa_K3308082 ( aka. CSP-BBa_K3308086 ) 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.

Results

We were unable to complete the second round Gibson Cloning in order to transform, grow, induce, and purify the protein.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 408
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 106
    Illegal BsaI.rc site found at 1613

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] Kimple, M. E., Brill, A. L., & Pasker, R. L. (2013). Overview of affinity tags for protein purification. Current protocols in protein science, 73, Unit–9.9. doi:10.1002/0471140864.ps0909s73

[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] Costa, S., Almeida, A., Castro, A., & Domingues, L. (2014). Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system. Frontiers in Microbiology, 5. doi: 10.3389/fmicb.2014.00063

[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] 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_K3308082. BBa_K3308083. BBa_K3308084. BBa_K3308085. BBa_K3308081. BBa_K3308087.

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