Difference between revisions of "Part:BBa K4016019"

 
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<partinfo>BBa_K4016019 short</partinfo>
 
<partinfo>BBa_K4016019 short</partinfo>
  
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This composite part is designed to generate GFP degradation with [[Part:BBa_K4016020]] (GFPnano - Pix D) through Pix E-Pix D interaction, GFPnano’s targeting function and Trim21 based ubiquitin-proteasome degradation system.
  
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<!-- Add more about the biology of this part here
+
 
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===Usage and Biology===
+
==Usage and Biology==
 +
Protein-protein interactions are powerful tools for manipulating biological processes, and opto-genetically - mediated induction of protein interactions has high spatial resolution without the need for exogenous cofactors. In this case, the dimer ”Pix D- Pix E” is introduced in our project in order to reach such function.
 +
 
 +
The two protein, Pix D and Pix E, can associate in the dark into large multi-subunit complexes that dissociate into dimers of Pix D and monomers of Pix E within seconds upon blue light stimulation. Fusing Pix D/Pix E fragment to the Trim21 and its targeting module, we can realize the Trim21-induced degradation on it’s target protein in the dark, and the Pix D-Pix E dissociation on blue light can induced the dissociation of Trim21 and its targeting module, so as to stop the degradation.
 +
 
 +
In our project, our composite part BBa_K4016019 is interacting with [[Part:BBa_K4016020]] (GFPnano - Pix D ), to specifically recognize and degrade GFP in the dark and stop the degradation in the light. If this can work, we can make an “OFF switch” in a system mediated by blue light.
 +
 
 +
 
 +
*Here is the mechanism of the system:
 +
 
 +
In the dark environment:
 +
 
 +
1.HA-Trim21-PixE connect with PixD-GFPnano through PixE-PixD interaction and forms a complex
 +
 
 +
2.GFPnano specifically recognize eGFP
 +
 
 +
3.eGFP is degraded by ubiquitin-proteasome system recruited by Trim21
 +
 
 +
In the blue light:
 +
 
 +
1. PixD-PixE dissociate
 +
 
 +
2. The degradation of eGFP stop because of the lack of trim21.
 +
 
 +
https://2021.igem.org/wiki/images/d/d9/T--NUDT_CHINA--Part_SchematicFigure_19-20.png
 +
Figure1. Schematic figure of BBa_K4016019 and BBa_K4016020
 +
 
 +
 
 +
==Characterization==
 +
This part is validated through four ways: enzyme cutting, PCR, Sequence, and functional testing.
 +
===PCR===
 +
The PCR is performed with Green Taq Mix by Vazyme.
 +
 
 +
F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTGCCACCATGGAGGTTCAACTGGAGGAGTCTG-3’
 +
 
 +
R-Prime: 5’- TGGATATCTGCAGAATTCTTACACGTTCACGCCGCCGTCGAT-3’
 +
 
 +
The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.
 +
 
 +
 
 +
===Enzyme Digestion===
 +
After the assembly the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The cutting procedure was performed with Hind III EcoR I restriction endonuclease bought.
 +
The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.
 +
 
 +
===Sequecing===
 +
The plasmid was sequenced correct.
  
 
<!-- -->
 
<!-- -->
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<span class='h3bb'>Sequence and Features</span>
+
===Sequence and Features===
 
<partinfo>BBa_K4016019 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4016019 SequenceAndFeatures</partinfo>
  
Line 17: Line 62:
 
<partinfo>BBa_K4016019 parameters</partinfo>
 
<partinfo>BBa_K4016019 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
==Functional test==
 +
This part (HA-Trim21-PixE) was tested together with BBa_K4016020 (GFPnano-PixD) and using they two respectively as control. (using eGFP~ Fluc-Rluc stable expressing HK293T cell line as follow, Rluc act as Internal reference)
 +
 +
===Method===
 +
*1.Cell transfection
 +
 +
(1)Seed HEK293T cells into 6-well cell culture plates.
 +
 +
(2)Culture for 16 h before transfection
 +
 +
(3)Total plasmid mixes of 800ng per well are mixed thoroughly in DMEM before a polyethylenimine (PEI) solution (1 mg/ml) is added into the plasmid mixture in a ratio of 1:5 (plasmid weight/PEI weight)
 +
 +
(4)The plasmid–PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for at least 6 h.
 +
 +
(5)Cells are then changed into fresh medium and culture for 18 h before subculture.
 +
 +
*2.Dual luciferase assay
 +
 +
(1)Wash HEK293T cells in 6-well plate with PBS and trypsinize prior to resuspension in fresh complete medium in a 15 ml microcentrifuge tube.
 +
 +
(2)Dispense 100ul of cell suspension (approximately 30000 cells per well) into 96 well plates.
 +
 +
(3)Capture the fluorescent image before apply blue light.(24h after transfection)
 +
 +
(4)Apply the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48/72 h before sampling and analysis assay. Capture the fluorescent image at 48/72 h respectively
 +
 +
(5)At 96h , add Reporter cell lysates in 96 well plates. (100uL per well)
 +
 +
(6)After extensive lysis, centrifugation at 10000-15000g for 3-5 min. Take the  supernatant for assay.
 +
 +
(7)Thaw firefly luciferase assay reagent and Renilla luciferase assay buffer, and bring to room temperature. Renilla luciferase assay substrate (100x) was placed on an ice bath or on an ice box for later use.
 +
 +
(8)Prepare Renilla luciferase assay working solution by adding Renilla luciferase assay substrate (100x) at 1:100 in an amount of 100 µ l per sample.
 +
 +
(9)Switch on the microplate reader, set the assay interval to 2 s and the assay time to 10 s.
 +
 +
(10)Take 20 to 100 ul of each sample for assay
 +
 +
(11)Add 100 ul of firefly luciferase assay reagent, measure the RLU (relative light unit) after mixing. Reporter cell lysate was used as a blank control.
 +
 +
(12)Add 100 ul of Renilla luciferase assay working solution
 +
 +
(13)The RLU value obtained from the Fluc assay was divided by the RLU value obtained from the Rluc assay. The degree of reporter gene activation of interest was compared between different samples according to the ratio obtained.
 +
 +
 +
 +
===Result===
 +
 +
 +
 +
===Reference===
 +
[1] Dine E, Gil AA, Uribe G, Brangwynne CP, Toettcher JE. Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli. Cell Syst. 2018 Jun 27;6(6):655-663.e5. doi: 10.1016/j.cels.2018.05.002. Epub 2018 May 30. PMID: 29859829; PMCID: PMC6023
 +
 +
[2] Yuan H, Bauer CE. PixE promotes dark oligomerization of the BLUF photoreceptor PixD. Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11715-9. doi: 10.1073/pnas.0802149105. Epub 2008 Aug 11. PMID: 18695243; PMCID: PMC2575306.
 +
 +
[3] Masuda S, Hasegawa K, Ishii A, Ono TA. Light-induced structural changes in a putative blue-light receptor with a novel FAD binding fold sensor of blue-light using FAD (BLUF); Slr1694 of synechocystis sp. PCC6803. Biochemistry. 2004 May 11;43(18):5304-13. d
 +
 +
[4] Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T, Hosouchi T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M, Tabata S. S
 +
 +
[5] Masuda S, Ono TA. Biochemical characterization of the major adenylyl cyclase, Cya1, in the cyanobacterium Synechocystis sp. PCC 6803. FEBS Lett. 2004 Nov 5;577(1-2):255-8. doi: 10.1016/j.febslet.2004.09.086. PMID: 15527795.
 +
 +
[6] Okajima K, Yoshihara S, Fukushima Y, Geng X, Katayama M, Higashi S, Watanabe M, Sato S, Tabata S, Shibata Y, Itoh S, Ikeuchi M. Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria. J Biochem. 200
 +
 +
[7] Sugimoto Y, Masuda S. In vivo localization and oligomerization of PixD and PixE for controlling phototaxis in the cyanobacterium Synechocystis sp. PCC 6803. J Gen Appl Microbiol. 2021 Jun 3;67(2):54-58. doi: 10.2323/jgam.2020.06.001. Epub 2020 Dec 21. PMID

Revision as of 01:30, 19 October 2021


HA-Trim21-PixE

This composite part is designed to generate GFP degradation with Part:BBa_K4016020 (GFPnano - Pix D) through Pix E-Pix D interaction, GFPnano’s targeting function and Trim21 based ubiquitin-proteasome degradation system.


Usage and Biology

Protein-protein interactions are powerful tools for manipulating biological processes, and opto-genetically - mediated induction of protein interactions has high spatial resolution without the need for exogenous cofactors. In this case, the dimer ”Pix D- Pix E” is introduced in our project in order to reach such function.

The two protein, Pix D and Pix E, can associate in the dark into large multi-subunit complexes that dissociate into dimers of Pix D and monomers of Pix E within seconds upon blue light stimulation. Fusing Pix D/Pix E fragment to the Trim21 and its targeting module, we can realize the Trim21-induced degradation on it’s target protein in the dark, and the Pix D-Pix E dissociation on blue light can induced the dissociation of Trim21 and its targeting module, so as to stop the degradation.

In our project, our composite part BBa_K4016019 is interacting with Part:BBa_K4016020 (GFPnano - Pix D ), to specifically recognize and degrade GFP in the dark and stop the degradation in the light. If this can work, we can make an “OFF switch” in a system mediated by blue light.


  • Here is the mechanism of the system:

In the dark environment:

1.HA-Trim21-PixE connect with PixD-GFPnano through PixE-PixD interaction and forms a complex

2.GFPnano specifically recognize eGFP

3.eGFP is degraded by ubiquitin-proteasome system recruited by Trim21

In the blue light:

1. PixD-PixE dissociate

2. The degradation of eGFP stop because of the lack of trim21.

T--NUDT_CHINA--Part_SchematicFigure_19-20.png Figure1. Schematic figure of BBa_K4016019 and BBa_K4016020


Characterization

This part is validated through four ways: enzyme cutting, PCR, Sequence, and functional testing.

PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime: 5’-CTAGCGTTTAAACTTAAGCTTGCCACCATGGAGGTTCAACTGGAGGAGTCTG-3’

R-Prime: 5’- TGGATATCTGCAGAATTCTTACACGTTCACGCCGCCGTCGAT-3’

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.


Enzyme Digestion

After the assembly the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The cutting procedure was performed with Hind III EcoR I restriction endonuclease bought. The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.

Sequecing

The plasmid was sequenced correct.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 204
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 873
    Illegal BamHI site found at 1411
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 161
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1251



Functional test

This part (HA-Trim21-PixE) was tested together with BBa_K4016020 (GFPnano-PixD) and using they two respectively as control. (using eGFP~ Fluc-Rluc stable expressing HK293T cell line as follow, Rluc act as Internal reference)

Method

  • 1.Cell transfection

(1)Seed HEK293T cells into 6-well cell culture plates.

(2)Culture for 16 h before transfection

(3)Total plasmid mixes of 800ng per well are mixed thoroughly in DMEM before a polyethylenimine (PEI) solution (1 mg/ml) is added into the plasmid mixture in a ratio of 1:5 (plasmid weight/PEI weight)

(4)The plasmid–PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for at least 6 h.

(5)Cells are then changed into fresh medium and culture for 18 h before subculture.

  • 2.Dual luciferase assay

(1)Wash HEK293T cells in 6-well plate with PBS and trypsinize prior to resuspension in fresh complete medium in a 15 ml microcentrifuge tube.

(2)Dispense 100ul of cell suspension (approximately 30000 cells per well) into 96 well plates.

(3)Capture the fluorescent image before apply blue light.(24h after transfection)

(4)Apply the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48/72 h before sampling and analysis assay. Capture the fluorescent image at 48/72 h respectively

(5)At 96h , add Reporter cell lysates in 96 well plates. (100uL per well)

(6)After extensive lysis, centrifugation at 10000-15000g for 3-5 min. Take the supernatant for assay.

(7)Thaw firefly luciferase assay reagent and Renilla luciferase assay buffer, and bring to room temperature. Renilla luciferase assay substrate (100x) was placed on an ice bath or on an ice box for later use.

(8)Prepare Renilla luciferase assay working solution by adding Renilla luciferase assay substrate (100x) at 1:100 in an amount of 100 µ l per sample.

(9)Switch on the microplate reader, set the assay interval to 2 s and the assay time to 10 s.

(10)Take 20 to 100 ul of each sample for assay

(11)Add 100 ul of firefly luciferase assay reagent, measure the RLU (relative light unit) after mixing. Reporter cell lysate was used as a blank control.

(12)Add 100 ul of Renilla luciferase assay working solution

(13)The RLU value obtained from the Fluc assay was divided by the RLU value obtained from the Rluc assay. The degree of reporter gene activation of interest was compared between different samples according to the ratio obtained.


Result

Reference

[1] Dine E, Gil AA, Uribe G, Brangwynne CP, Toettcher JE. Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli. Cell Syst. 2018 Jun 27;6(6):655-663.e5. doi: 10.1016/j.cels.2018.05.002. Epub 2018 May 30. PMID: 29859829; PMCID: PMC6023

[2] Yuan H, Bauer CE. PixE promotes dark oligomerization of the BLUF photoreceptor PixD. Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11715-9. doi: 10.1073/pnas.0802149105. Epub 2008 Aug 11. PMID: 18695243; PMCID: PMC2575306.

[3] Masuda S, Hasegawa K, Ishii A, Ono TA. Light-induced structural changes in a putative blue-light receptor with a novel FAD binding fold sensor of blue-light using FAD (BLUF); Slr1694 of synechocystis sp. PCC6803. Biochemistry. 2004 May 11;43(18):5304-13. d

[4] Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T, Hosouchi T, Matsuno A, Muraki A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada M, Yasuda M, Tabata S. S

[5] Masuda S, Ono TA. Biochemical characterization of the major adenylyl cyclase, Cya1, in the cyanobacterium Synechocystis sp. PCC 6803. FEBS Lett. 2004 Nov 5;577(1-2):255-8. doi: 10.1016/j.febslet.2004.09.086. PMID: 15527795.

[6] Okajima K, Yoshihara S, Fukushima Y, Geng X, Katayama M, Higashi S, Watanabe M, Sato S, Tabata S, Shibata Y, Itoh S, Ikeuchi M. Biochemical and functional characterization of BLUF-type flavin-binding proteins of two species of cyanobacteria. J Biochem. 200

[7] Sugimoto Y, Masuda S. In vivo localization and oligomerization of PixD and PixE for controlling phototaxis in the cyanobacterium Synechocystis sp. PCC 6803. J Gen Appl Microbiol. 2021 Jun 3;67(2):54-58. doi: 10.2323/jgam.2020.06.001. Epub 2020 Dec 21. PMID