Difference between revisions of "Part:BBa K3308085"

 
 
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3308085 short</partinfo>
 
<partinfo>BBa_K3308085 short</partinfo>
 +
===NSP-construct===
 +
===Overview===
 +
[[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]]]
  
−
nsp
+
[[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 full N-terminal of NrdJ-1. We have denoted it as the NSP construct. This costruct acts a positive control of splicing of gp41-1]]
  
−
<!-- Add more about the biology of this part here
+
===Design===
−
===Usage and Biology===
+
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, GTNPC, the same as BBa_K33080081.[2,3,4]
 +
This part is the expected product of functional splicing of gp41-1 intein .[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. This part also had the same design consideration for insertion of a flanking sequence between the extein the NrdJ-1 N intein in part BBa_K3308081 .[2]
 +
 
 +
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.[[#References|[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. [[#References|[2]]]
 +
 
 +
 
 +
===Usage===
 +
The main purpose of this part was to act as apositive control for splicing of composite parts BBa_K3308081 and BBa_K3308082 and also a functional N-intein NrdJ-1 that should be able to splice with the spliced product of BBa_K3308083 and BBa_K3308084 ( 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. We would have epected the addition of MBP to this construct specifically because it contains a C-terminal intein gp41-1 C, would have increased solubility.
  
−
<!-- -->
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3308085 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3308085 SequenceAndFeatures</partinfo>
  
 +
===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.
  
−
<!-- Uncomment this to enable Functional Parameter display
+
This part is this set of nested Inteins constructs:
−
===Functional Parameters===
+
<partinfo>BBa_K3308082</partinfo>.
−
<partinfo>BBa_K3308085 parameters</partinfo>
+
<partinfo>BBa_K3308083</partinfo>.
−
<!-- -->
+
<partinfo>BBa_K3308084</partinfo>.
 +
<partinfo>BBa_K3308081</partinfo>.
 +
<partinfo>BBa_K3308086</partinfo>.
 +
<partinfo>BBa_K3308087</partinfo>.

Latest revision as of 02:03, 21 October 2019


GB1-GTNPC-[NrdJ-1 N (1-4)]-[NrdJ-1 N (5-104)]

NSP-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 full N-terminal of NrdJ-1. We have denoted it as the NSP construct. This costruct acts a positive control of splicing of gp41-1

Design

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, GTNPC, the same as BBa_K33080081.[2,3,4] This part is the expected product of functional splicing of gp41-1 intein .[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. This part also had the same design consideration for insertion of a flanking sequence between the extein the NrdJ-1 N intein in part BBa_K3308081 .[2]

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

The main purpose of this part was to act as apositive control for splicing of composite parts BBa_K3308081 and BBa_K3308082 and also a functional N-intein NrdJ-1 that should be able to splice with the spliced product of BBa_K3308083 and BBa_K3308084 ( 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. We would have epected the addition of MBP to this construct specifically because it contains a C-terminal intein gp41-1 C, would have increased solubility.

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_K3308081. BBa_K3308086. BBa_K3308087.