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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Pichia pastoris GS115,E.coli DH5α,plasmid pPIC9K
PrimeSTAR HS DNA Polymerase,5×PrimeSTAR Buffer(Mg2+ plus),dNTP Mixture,OMEGA Plasmid Mini Kit,OMEGA Gel Extraction Kit,T4 DNA ligase,restriction enzyme Not I,Ecor I,SaI I,Quickcut buffer,agarose,Electrophoresis buffer,Maker DS5000,10 x Gel-loading buffer,1x TAE,BCA Protein Assay Kit,In-Fusion HD cloning system,Tris-HCl,SDS,Acrylamide,Methylene,Ammonium Persulfate,Trizma,Glycine,Glycerol,ß Mercapto Ethanol,Bromo Phenol Blue,TEMED,Methanol CP,Acetic Acid CP,Coomasie Brilliant Blue R.
LB medium,YPD medium,ampicillin.
Protein expression
PCR amplification:
With appropriate pairs of primers,PCR was carried out to prepare ABF2,PP2CA and SnRK2.2 segments.
Plasmind construction:
ABF2,PP2CA,SnRK2.2 coding sequences were cloned into cloning&expression vector pPIC9K after PCR amplification by means of restriction enzyme digestion and DNA ligation.
Fig1: Protein expression plasmids
Transformation and protein collection：
pPIC9K-ABF2,pPIC9K-PP2CA and pPIC9K-SnRK2.2 were transformed into Pichia pastoris GS115 cells.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 these three proteins. see protein expression results
Bi-stable function
PCR amplification:
Using appropriate pairs of primers with overlaps to fulfill the requirements of In-Fusion,PCR was carried out to prepare TTADH1,PP2CA,pCyc,SnRK2.2,ABF2-TTADH1,pRD29A-Kozak and GFP segments
Plasmid construction:
Construct pSB1C3-PP2CA-TTADH1-pCyc, pSB1C3-Kozak-SnRK2.2-TTADH1-pCyc, pSB1C3-Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP by In-fusion HD cloning system as the first round. Then collect the -PP2CA-TTADH1-pCyc, -Kozak-SnRK2.2-TTADH1-pCyc, Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP these three segments into the second round In-fusion to build pPIC9K-PP2CA-TTADH1-pCyc-Kozak-SnrK2.2-TTADH1-pCyc-Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP-LVAtag functional circuit.
Fig2:Eukaryotic bi-stable function characterized plasmid assembly work flow
At the same time,we employ 3A assembly to get control group pPIC9k--pCyc-Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP.
Fig3: Eukaryotic bi-stable function control plasmid
Transformation and induction:
pPIC9K-PP2CA-TTADH1-pCyc-Kozak-SnRK2.2-TTADH01-pCyc-Kozak-ABF2-TTADH1-pRD29A-Kozak-GFP and its control group were transformed into Pichia pastoris GS115 cells.Then they were grown in YPD medium containing 1‰ ampicillin.Methanol acts as an inducer to control the transcription of gene PP2CA. Without PP2CA,this circuit was to test the ability for SnRK2.2 to phosphorylate ABF2.
Fluorescence detection:
Microplate spectrophotometer was applied to detect and quantify GFP expression.The strain transformed with the whole circuit and its control are set to verify SnRK2.2’s function.Moreover,comparing with the absence of methanol induction,the whole circuit containing strain induced by methanol can prove PP2CA’s function when there is GFP fluorescence intensity decrease. Each circuit was tested with 3 parallels. see Bi-stable function results
Fig4：ideal 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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Media and antibiotics:
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E.coli DH5α,plasmid pSB1C3,plasmid PETDuet-1
PrimeSTAR HS DNA Polymerase,5×PrimeSTAR Buffer(Mg2+ plus),dNTP Mixture,OMEGA Plasmid Mini Kit,OMEGA Gel Extraction Kit,T4 DNA ligase、restriction enzyme Xba l,Ecor I,Spe I,Pst I,Quickcut buffer,agarose,Electrophoresis buffer,Maker DS5000,10 x Gel-loading buffer,1x TAE,BCA Protein Assay Kit,In-Fusion HD cloning system
LB medium,ampicillin,chloramphenicol
Flexstation 3 plate reader
Protein and protein interaction
PCR
With appropriate pairs of primers,PCR was carried out to prepare CII,CIII,CII-TT,pRE-RBS and GFP-LVAtag segments.CII-TT and pRE-RBS were amplified using primers with overlaps to fulfill the requirements of In-Fusion.
Plasmid construction
pSB1C3-CII-TT-pRE-RBS-GFP was constructed by In-Fusion cloning method, at the same time, 3A assembly was applied to construct plasmid CIII-RBS-CIII-pSB1C3, CII-RBS-CII-pSB1C3 and futher pSB1C3-CIII-RBS-CIII-RBS-CII-pRE-RBS-GFP-tag and pSB1C3-CII-RBS-CII-RBS-CII-pRE-RBS-GFP-tag . Finally, transfer the backbone of the circuit segments to gain PETDuet-1--CIII-RBS-CIII-RBS-CII-pRE-RBS-GFP-tag and PETDuet-1--CII-RBS-CII-RBS-CII-pRE-RBS-GFP-tag.
Fig5: Protein&protein interaction characterized plasmid assembly work flow
Transformation and induction
All plasmids and their control groups were transformed into E.coli DH5α cells. Then they were grown in LB medium containing 1‰ ampicillin. IPTG acts as an inducer to activate T7 promoter already existed in PETDuet-1 backbone.
Fluorescence detection
Microplate spectrophotometer was applied to detect and quantify GFP expression. Comparing three-CIII tandem circuit to three-CII tandem, we can verify that if CIII can inhibit Ftsh degrading CII. see the protein&protein reaction results
Fig6: ideal CIII&Ftsh characterization result
Protein and promoter interaction
Three pairs of protein and promoter were examined to confirm the property of their interactions.
PCR and plasmid construction
CII and pRE: With appropriate pairs of primers,PCR was carried out to prepare CII,CII-TT,pRE-RBS and GFP segments. PETDuet-1--CII-TT-pRE-RBS-GFP was constructed by In-Fusion cloning method and PETDuet-1--pRE-RBS-GFP was successively constructed through enzyme digestion and ligation to serve as control group.
Fig7: CII&pRE interaction characterization plasmid and its control
CI and pR: With appropriate pairs of primers,PCR was carried out to prepare CI-TT,pR-RBS and GFP-LAVtag segments. PETDuet-1--CI-TT-pR-RBS-GFP-LAVtag was constructed by In-Fusion cloning method and then its control group PETDuet-1--pR-RBS-GFP-LAVtag was constructed the same way as CII and pRE’s.
Fig8:CI&pR interaction characterization plasmid and its control
CI and pR:The same as above.
Fig9:Cro&pRM interaction characterization plasmid and its control
Transformation and induction
All plasmids and their control groups were transformed into E.coli DH5α cells.Then they were grown in LB medium containing 1‰ ampicillin. IPTG acts as an inducer to activate T7 promoter already existed in 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. With increasing CII’s tandem expressing number ( PETDuet-1--CII-RBS-CII-RBS-CII-TT-pRE-RBS-GFP,PETDuet-1--CII-TT-pRE-RBS-GFP), we can prove that CII serves as transcriptional factor if GFP fluorescence intensity goes up. see the Protein&promoter results
Fig10: ideal CII&pRE characterization result
PETDuet-1--CI-TT-pR-RBS-GFP-LAVtag was constructed to verify CI’s ability to block pR. Comparing to circuit of control group PETDuet-1--pR-RBS-GFP-LAVtag, we can prove CI’s function if GFP fluorescence intensity goes down when induced by IPTG.see the Protein&promoter results
Fig11: ideal CI&pR characterization result
Cro and pRM are characterized the same way..see the Protein&promoter results
Fig12: ideal Cro&pRM characterization result
Tri-stable function
PCR
With appropriate pairs of primers,PCR was carried out to prepare segments from circuits constructed above (Protein&protein interaction characterization circuits and Protein&promoter characterization circuits).
Plasmid construction
PETDuet-1--pRE-RBS-Cro-RBS-CII-TT-prtp-CI-TT-pR-RBS-CIII-RFP-TT-pRM-RBS-GFP was constructed with help of both In-Fusion HD cloning system and 3A assembly.
Fig13: tri-stable function characterization plasmind
Transformation and induction
PETDuet-1--pRE-RBS-Cro-CII-TT-prtp-CI-TT-pR-RBS-CIII-RFP-TT-pRM-RBS-GFP was transformed into E.coli DH5α cells.Then they were cultivated 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 achieve two stable expression state:GFP expressing state when induced by IAA and RFP by IPTG. see Tri-stable function results
Fig14: ideal tri-stable characterization result
Application circuit construction
Materials: The same as Prokaryote’s
lactic acid banlance function:
PCR
With appropriate pairs of primers,PCR was carried out to prepare segments placm-pRE-RBS, Cro, RBS-CII-TT, patp2-RBS, CI-TT, pR-RBS-CIII, RBS-iLDH-TT, pRM-RBS-beta-gala (partially synthesized by lDT)
Plasmid construction
pSB1C3-placm-pRE-RBS-Cro-RBS-CII-TT, pSB1C3-patp2-RBS-CI-TT-pR-RBS-CIII, pSB1C3- RBS-iLDH-TT-pRM-RBS-beta-gala was constructed through In-Fusion HD cloning system. Then we employ 3A assembly to construct placm-pRE-RBS-Cro-RBS-CII-TT-Patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-gala circuit and transfer backbone from pSSB1C3 to PETDuet-1.
Fig15: lactic acid acid balance functional plasmid
Transformation
PETDuet-1--placm-pRE-RBS-Cro-RBS-CII-TT-Patp2-RBS-CI-TT-pR-RBS-CIII-RBS-iLDH-TT-pRM-RBS-beta-gala was transformed into E.coli DH5α cells. 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.
