Difference between revisions of "Part:BBa K3308087"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3308087 short</partinfo>
 
<partinfo>BBa_K3308087 short</partinfo>
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===MSP-construct===
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===Overview===
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[[File:Coded- Nested intein diagram.png|300px|thumb|right| ]] 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. [[#References|[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.[[#References|[1]]]
  
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msp + MBP
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[[File:pvs45- 51 general diagram.png|900px|thumb|center|'''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. [[#References|[3]]]. This composite part contains the N-terminal of primary splicing intein, gp41-1. We have denoted it as the MSP construct. This costruct is the positive control of functional splciign fo NrdJ-1. If there is SPlicing between NSP and CSP then the Mixed spliced product (this part) would be constructed.]]
  
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<!-- Add more about the biology of this part here
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===Design===
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===Usage and Biology===
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After expression and attempted purification of <partinfo>BBa_K3308007</partinfo>, We decided that we would conduct Gibson Assembly of the part into a different plasmid backbone <partinfo>BBa_K3308093</partinfo> consisting of Maltose Binding Protein[[#References|[3,5]]].  Maltose Bidning Protein is a relatively large 42 kDa
 +
This composite part contains the Full extein sequence, The N and C terminal Exteins, GB1 and gpD
 +
We have denoted it as the NSP construct. This costruct acts a positive control of splicing of gp41-1 (BBa_K3308081 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 and gp41-1. inteins which will brign together functional splicing of external NrdJ-1 intein.
 +
 
 +
===Usage===
 +
The main purpose of this part was to act as a positive control for splicing of composite parts BBa_K3308011 and BBa_K3308012
 +
In the test of the full nested intein system when N1, N2, C1, and C2 are all added together sequentially NSP and CSP should be made, then those splicign will in turn produce this composite part MSP
 +
 
 +
N1 BBa_K3308081
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N2 BBa_K3308082
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NSP BBa_K3308085
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C1 BBa_K3308083
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C2 BBa_K3308084
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CSP BBa_K3308086
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 +
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.
  
 
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<partinfo>BBa_K3308087 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3308087 SequenceAndFeatures</partinfo>
  
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===References===
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[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
  
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<!-- Uncomment this to enable Functional Parameter display
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[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
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===Functional Parameters===
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<partinfo>BBa_K3308087 parameters</partinfo>
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[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
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<!-- -->
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 +
[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===
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This page was was last updated by Pittsburgh 2019 team.
 +
 
 +
This part is this set of nested Inteins constructs:
 +
<partinfo>BBa_K3308082</partinfo>.
 +
<partinfo>BBa_K3308083</partinfo>.
 +
<partinfo>BBa_K3308084</partinfo>.
 +
<partinfo>BBa_K3308085</partinfo>.
 +
<partinfo>BBa_K3308086</partinfo>.
 +
<partinfo>BBa_K3308081</partinfo>.

Latest revision as of 02:21, 21 October 2019


GB1-GTNPC-SEIVL-gpD

MSP-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 N-terminal of primary splicing intein, gp41-1. We have denoted it as the MSP construct. This costruct is the positive control of functional splciign fo NrdJ-1. If there is SPlicing between NSP and CSP then the Mixed spliced product (this part) would be constructed.

Design

After expression and attempted purification of BBa_K3308007, 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 Full extein sequence, The N and C terminal Exteins, GB1 and gpD We have denoted it as the NSP construct. This costruct acts a positive control of splicing of gp41-1 (BBa_K3308081 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 and gp41-1. inteins which will brign together functional splicing of external NrdJ-1 intein.

Usage

The main purpose of this part was to act as a positive control for splicing of composite parts BBa_K3308011 and BBa_K3308012 In the test of the full nested intein system when N1, N2, C1, and C2 are all added together sequentially NSP and CSP should be made, then those splicign will in turn produce this composite part MSP

N1 BBa_K3308081 N2 BBa_K3308082 NSP BBa_K3308085 C1 BBa_K3308083 C2 BBa_K3308084 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.

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_K3308084. BBa_K3308085. BBa_K3308086. BBa_K3308081.