Eukaryote
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Materials
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Strains and vectors:
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Enzymes and reagents:
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Media and Antibiotics:
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Equipment:
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Pichia pastoris GS115, E.coli DH5α, plasmid pPIC9K
PrimeSTAR HS DNA Polymerase（Takara R010A）, 5×PrimeSTAR Buffer(Mg2+ plus) （Takara R010A）, dNTP Mixture（Takara R010A）, OMEGA Plasmid Mini Kit（Omega D6943-02）, OMEGA Gel Extraction Kit（Omega D2500-02）, T4 DNA ligase（Takara 2011A）, restriction enzyme and Quickcut buffer Not I（Takara 1623）, EcoR I（Takara 1611）, SaI I（Takara 1636）, Spe I (Takara 1631), Xba I (Takara 1634), Agarose（Biowest A0009-100）, Maker DS5000（DongSheng M1111）, 10 x Gel-loading buffer（Takara 9157）, 1x TAE buffer（Sangon B548101-0500）, BCA Protein Assay Kit（Takara T9300A）, In-Fusion HD cloning system（Clontech 639648）, Tris-HCl/SDS (Sangon B546021-0250&Sangon B546022-0250）, Acrylamide（Sangon A100341-0100）, N,N’-Methylenebisacrylamide（Sangon A100172-0025）, Ammonium Persulfate（Sangon A100486-0100）, Glycine（Sangon A100167-0500）, Glycerol（Sangon A100854-0500）, ß Mercapto Ethanol, Bromo Phenol Blue, TEMED（Sangon A100761-0100）, Methanol（Sangon A506806-0500）, Acetic Acid（Sangon A501931-0500）, Coomasie Brilliant Blue R（Sangon A100472-0025）.
LB medium, YPD medium, Ampicillin（Sangon A100741-0025）
Flexstation 3 plate reader
Protein Expression
PCR Amplification:
With appropriate primers, PCR was carried out to amplify target gene ABF2, PP2CA and SnRK2.2.
Plasmid Construction:
Coding sequences ABF2, PP2CA and SnRK2.2 were cloned into cloning&expression vector pPiC9K by restriction enzyme digestion and DNA ligation.
Fig 1: Protein Expression Plasmids
Transformation and Protein Collection：
Plasmid ABF2-pPIC9K, PP2CA-pPIC9K and SnRK2.2-pPIC9K were transformed into Pichia pastoris GS115 cells by Electroporation.Then they were grown in YPD medium containing 1‰ ampicillin. Protein ABF2, PP2CA and SnRK2.2 were purified and collected during the process of protein ultrafiltration.
Verification and Quantification:
SDS-PAGE and BCA protein assay were performed to verify and quantify three proteins mentioned above.
Bi-stable Function Characterization
PCR Amplification:
Using appropriate primers with overlaps to fulfill the requirements of In-Fusion, PCR was carried out to amplify target gene TTADH1, PP2CA, pCyc, SnRK2.2, ABF2-TTADH1, pRD29A-Kozak and GFP.
Plasmid Construction:
At first, plasmids PP2CA-TTADH1-pCyc-pSB1C3, Kozak-SnRK2.2-TTADH1-pCyc-pSB1C3, Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP-pSB1C3 were constructed by restriction enzyme digestion and In-fusion HD cloning system. Then the PP2CA-TTADH1-pCyc, Kozak-SnRK2.2-TTADH1-pCyc, Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP fragments were used in the second round In-fusion to build the function circuit, BBaK30-pPIC9K. At the same time, 3A assembly was employed to get the control plasmid Control-pPIC9k.
Fig 2: Eukaryotic bi-stable function characterized plasmid assembly work flow
| Part Number | Description |
|---|---|
| BBa_K2036030 | PP2CA-TTADH1-pCyc-Kozak-Snrk2.2-TTADH1-pCyc-Kozak-ABF2-TTADH1-prd29A-Kozak-GFP-LVAssrAtag |
Fig 3: Eukaryotic bi-stable function control plasmid
Transformation and Induction:
Plasmid BBaK30-pPIC9K and its control one were transformed into Pichia pastoris GS115 cells. Then they were grown in YPD medium containing 1‰ ampicillin. Methanol acts as the inducer to control the transcription of gene PP2CA. The control circuit was designed to test the ability for SnRK2.2 to phosphorylate ABF2 without PP2CA.
Fluorescence Detection:
Microplate spectrophotometer was applied to detect and quantify the expression of GFP. The strains transformed with whole circuits and their control ones are set to characterize the SnRK2.2’s function. To prove PP2CA’s function, strain containing the whole circuit will decrease GFP fluorescence intensity under the induction of methanol. Each circuit was tested with 3 parallels.
Fig 4：Proposed GFP Fluorescence detection result
Prokaryote
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Materials
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Strains and Vectors:
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Enzymes and Reagents:
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Media and Antibiotics:
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Equipment:
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E.coli DH5α, plasmid pSB1C3, plasmid PETDuet-1
PrimeSTAR HS DNA Polymerase（Takara R010A）, 5×PrimeSTAR Buffer(Mg2+ plus) （Takara R010A）, dNTP Mixture（Takara R010A）, OMEGA Plasmid Mini Kit（Omega D6943-02）, OMEGA Gel Extraction Kit（Omega D2500-02）, T4 DNA ligase（Takara 2011A）, restriction enzyme and Quickcut buffer Not I（Takara 1623）, EcoR I（Takara 1611）, SaI I（Takara 1636）, Spe I (Takara 1631), Xba I (Takara 1634), Agarose（Biowest A0009-100）, Maker DS5000（DongSheng M1111）, 10 x Gel-loading buffer（Takara 9157）, 1x TAE buffer（Sangon B548101-0500）, BCA Protein Assay Kit（Takara T9300A）, In-Fusion HD cloning system（Clontech 639648）
LB medium, Ampicillin（Sangon A100741-0025）, Chloramphenicol（Sangon A100230-0025）
Flexstation 3 plate reader
Protein and Protein Interaction
PCR:
With appropriate primers, PCR was carried out to amplify target gene CII, CIII, CII-TT, pRE-RBS and GFP-LVAtag. CII-TT and pRE-RBS were amplified using primers with overlaps to fulfill the requirements of In-Fusion.
Plasmid Construction:
Plasmid CII-TT-pRE-RBS-GFP-pSB1C3 was constructed by In-Fusion cloning method, at the same time, 3A assembly were applied to construct plasmid CIII-RBS-CIII-pSB1C3, CII-RBS-CII-pSB1C3, BBaK14-pSB1C3 and BBaK15-pSB1C3. Finally, the constructed circuits were transferred into another backbone PETDuet-1 to gain BBaK14-PETDuet-1 and BBaK15-PETDuet-1.
Fig 5: Protein and protein interaction characterized plasmid assembly work flow
| Part Number | Description |
|---|---|
| BBa_K2036014 | RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag |
| BBa_K2036015 | RBS-CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag |
Transformation and Induction:
All plasmids and their control groups were transformed into E.coli DH5α cells by standard competent cell transformation method. Then they were grown in LB medium containing 1‰ ampicillin. IPTG works as the inducer to activate T7 promoter in the PETDuet-1 backbone.
Fluorescence Detection:
Microplate spectrophotometer was applied to detect and quantify GFP expression.
Comparing the three-CIII tandem circuit with the three-CII tandem one, we can verify that whether CIII can inhibit Ftsh’s degradation of CII.
Fig 6: Proposed CIII&Ftsh characterization result
Protein and Promoter Interaction
Three pairs of proteins and promoters were examined to confirm the property of their interactions.
PCR and Plasmid Construction:
CII and pRE: With appropriate primers, PCR was carried out to amplify CII, CII-TT, pRE-RBS and GFP. BBaK32-PETDuet-1 was constructed by In-Fusion cloning method and BBaK11-PETDuet-1 were successively constructed through enzyme digestion and ligation to serve as control group.
Fig 7: CII&pRE interaction characterization plasmid and its control
| Part Number | Description |
|---|---|
| BBa_K2036032 | CII-TT-pRE-RBS-GFP-LVAssrAtag |
| BBa_K2036011 | pRE-GFP-LVAssrAtag |
CI and pR: With appropriate primers, PCR was carried out to amplify CI-TT, pR-RBS and GFP-LVAtag. BBaK16-PETDuet-1 was constructed by In-Fusion cloning method and then it’s’ control group BBaK17-PETDuet-1, CII and pRE plasmids were constructed in the same way.
Fig 8: CI&pR interaction characterization plasmid and its control
| Part Number | Description |
|---|---|
| BBa_K2036016 | pR-GFP-LVAssrAtag |
| BBa_K2036017 | CI-TT-pR-RBS-GFP-LVAssrAtag |
Cro and pRM:The same method as above.
Fig 9: Cro&pRM interaction characterization plasmid and its control
| Part Number | Description |
|---|---|
| BBa_K2036009 | pRM-GFP-LVAssrAtag |
| BBa_K2036010 | Cro-TT-pRM-RBS-GFP-LVAssrAtag |
Transformation and Induction:
All plasmids and their control groups were transformed into E.coli DH5α cells by standard competent cell transformation method.Then they were grown in LB medium containing 1‰ ampicillin. IPTG acts as the inducer to activate T7 promoter in the PETDuet-1 backbone.
Fluorescence Detection:
Microplate spectrophotometer was applied to detect and quantify GFP expression. CII was overexpressed to eliminate the interruption of constitutive expressed FtsH. We can prove that CII serves as a transcriptional factor if GFP fluorescence intensity rises with the increasing of the number of CII repeats (BBaK15-PETDuet-1, BBaK32-PETDuet-1).
Fig 10: Proposed CII&pRE characterization result
BBaK17-PETDuet-1 was constructed to verify CI’s ability to block pR. Comparing to circuit of control group pR-RBS-GFP-LVAtag-PETDuet-1, we can prove CI’s function if GFP fluorescence intensity goes down when induced by IPTG.
Fig 11: Proposed CI&pR characterization result
Cro and pRM are characterized in the same way.
Fig 12: Proposed Cro&pRM characterization result
Tri-stable function
PCR:
With appropriate primers, PCR was carried out to amplify fragments from circuits constructed above (Protein and protein interaction characterization circuits and Protein and promoter characterization circuits).
Plasmid Construction:
Plasmid BBaK27-PETDuet-1 was constructed using both In-Fusion HD cloning system and 3A assembly.
Fig 13: Tri-stable function characterization plasmid
| Part Number | Description |
|---|---|
| BBa_K2036027 | pRE-RBS-Cro-RBS-CII-TT-ptrp-RBS-CI-TT-pR-RBS-CIII-RBS-RFP-LAAssrAtag-TT-pRM-RBS-GFP-LVAssrAtag |
Transformation and Induction:
BBaK27-PETDuet-1 was transformed into E.coli DH5α cells by standard competent cell transformation method. Then they were grown in LB medium containing 1‰ ampicillin. T7 RNA Polymerase can initiate T7 promoter induced by IPTG.
Fluorescence Detection:
Microplate spectrophotometer was applied to detect and quantify GFP and RFP expression. If successful, it will occur two stable expression states: GFP expressing state when induced by IAA and RFP by IPTG.
Fig 14: Proposed tri-stable characterization result
Application Circuit Construction
Materials: The same as Prokaryote’s
Lactic Acid Balance Function:
PCR:
With appropriate primers, PCR was carried out to amplify genes placm-pRE-RBS, Cro, RBS-CII-TT, patp2-RBS, CI-TT, pR-RBS-CIII, RBS-iLDH-TT and pRM-RBS-beta-galactosidase (partially synthesized by lDT)
Plasmid Construction:
Plasmid BBaK28-pSB1C3 were constructed by In-Fusion HD cloning system. Then we employ 3A assembly to construct BBaK28-PETDuet-1.
Fig 15: lactic acid balance functional plasmid
| Part Number | Description |
|---|---|
| BBa_K2036028 | placm-pRE-RBS-Cro-RBS-CII-TT-patp2-RBS-CI-TT-pR-RBS-CIII-RBS-ompA-iLDH-TT-pRM-RBS-beta-galactosidase |
Transformation:
BBaK28-PETDuet-1 was transformed into E.coli DH5α cells by standard competent cell transformation method. Then they were grown in LB medium containing 1‰ ampicillin.
Lactic Acid Balance Function:
Enzyme activity assay is applied to detect the tri-stable function in vitro. We will simulate the intestine micro-environment and intermittently add lactose as patients’ normal milk intake. It is supposed to keep lactic acid level in balance on account of iLDH activity and our designed positive feedback circuit.
