Part:BBa_K3308083

[NrdJ-1 C (1-4)]-[Tvo VMA C48 N]

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 C1 construct. The N-Terminus of this split intein is fused to a split site we have chosen in the C-terminus of the intein NrdJ-1. In order to choose this split site we had to consider that the N-terminal flanking sequences for TvoVMA inteins.

Design

After expression and attempted purification of BBa_K3308009, 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 N terminus of TvoVMA Intein, it is covalently attached to the first half of the C-terminal NrdJ-1 InteinBBa_K3308070. The main purpose of this construct is to preserve functional splicing of TvoVma N intein.[1]

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_K3308084, 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]

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_K3308084 C2 to form the spliced product, the full terminus of the N- NrdJ-1 Intein CSP BBa_K3308086.

Results

Because this part was only the first few amino acids of the NrdJ-1 C terminal (due to the fact that we chose a split site that was closer to the N terminus of the C- Intein NrdJ-1), the construct did not as much of an issue purifying.

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 30 corresponds with this composite part. There is some level of protein the elution; however, we cannot deduce the actual concentration of our protein of interest

There was clear band in the elution which matched the expected kDa size of the contruct. The elution from the purification step was then dialyzed into a splicing buffer in which splicing reactions could be performed. The results of diafiltration are seen in the last two lanes of Figure 3. The band density decreases, however, this was enough for us to see splicing reaction with it's compliment. Because the compliment of the TvoVMA intein was hard to purify without the presence of maltose binding protein, we decided to combine this composite part and BBa_K3308084. This reaction was were promising because we deducted that there was splicing between the TvoVma inteins when both constructs had MBP. The results of this splicing reaction will be posted in our notebook page. We have also tried to combine this construct with the TvoVMA

Figure 3: On this gel we tested diafiltration of pVS30(BBa_K3308010) in the last two lanes The above gel is a SDS-PAGE of the last step of purification E- Elution comapred to after the elution was dialyzed

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 1227
  Illegal SapI.rc site found at 1527
  

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_K3308086. BBa_K3308087.