Part:BBa_K3308084

[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 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 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

After failed efficient expression and purification of BBa_K3308010, 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 construct has the first half of the N-terminal NrdJ-1 Intein, which means that it is covalently attached to the N-terminal extein, GB1BBa_K3308065. 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_K3308083, to form fully fucntional C-terminus NrdJ-1 (BBa_K3308086). If splicing occurs as planned between these two constructs then, addition of BBa_K3308083, BBa_K3308084, and BBa_K3308085 should result in the effective reconstruction of the full extein (GB1-GTNPC-SEIVL-gpD)- BBa_K3308087

The N1 position, which corresponds to the last amino acid of NrdJ-1 did not match the original sequence reported to preserve functionality in have been predicted to need in order to preserve splicing. The N-2 amino acid was a Glycine so we assumed that the N-2 properties as EWG or EDG were not as important for this intein.[7] The N-1 intein was a Lysine so we thought that this particular intein might need the resonance and hindrance of this amino acid, however this site has been changed before and able to still produce splicing. When looking into the block B site of gp41-1, we saw that there were many acid and base amino acid since this block is involved in the first step as reviewed above we thought that it would be more important than the N-1 intein here. [2]

Considering these things and the fact that any percent yield would still be considered a success for our current methods we decided that this site would be a strong candidate for success.

Usage

ach 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_K33080083 C2 to form the spliced product, the full terminus of the N- NrdJ-1 Intein CSP BBa_K3308086. Because purification did not look much better with the addition of MBP, we decided to run lysate tests that would allow TvoVMA trnaslated in the cells to interact and splice.

Results

File:Purification 47 48 lysate.png

Figure 2: The SDS-Page gel above is a lysate mixture test combing lysate mictures of 47(BBa_K3308083 and 48 BBa_K3308084

In the very last column we cna see a clear]]

File:Purification 47 48 lysate.png

Figure 2: The SDS-Page gel above is a lysate mixture test combing lysate mictures of 47(BBa_K3308083 and 48 BBa_K3308084

In the very last column we cna see a clear]]

The following construct was tested for splicing of TvoVMA by combining lysates of this construct and BBa_K3308083. We were able to see efficient splicing of these two constructs and conclude the Nested C terminal NrdJ-1 was spliced together. We can clearly see the starting concentration of the two constructs, the depletion of of the starting intein constructs, and formation of the spliced product which is smaller in size as well.

ADD the PICTURE and description

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
  

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_K3308081. BBa_K3308085. BBa_K3308086. BBa_K3308087.