Difference between revisions of "Team:HUST-China/Experiments"

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                 <h4>Verification and quantification:</h4>
 
                 <h4>Verification and quantification:</h4>
 
                 <!-- <img src="" alt=""> -->
 
                 <!-- <img src="" alt=""> -->
−
                 <p>SDS-PAGE and BCA protein assay were performed to verify and quantify these three proteins. <a href="">see protein expression results</a>
+
                 <p>SDS-PAGE and BCA protein assay were performed to verify and quantify these three proteins. <a href="https://2016.igem.org/Team:HUST-China/Results#result_proex">see the protein expression result</a>
 
                 </p>
 
                 </p>
 
                 <h3>Bi-stable function</h3>
 
                 <h3>Bi-stable function</h3>
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                 <h4>Fluorescence detection:</h4>
 
                 <h4>Fluorescence detection:</h4>
 
                 <p>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.
 
                 <p>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.
−
                     <a href="">see Bi-stable function results</a></p>
+
                     <a href="">see the results</a></p>
 
                 <img src="https://static.igem.org/mediawiki/2016/e/eb/T--HUST-China--Experiments-Fig4.png" alt="" class="img-responsive">
 
                 <img src="https://static.igem.org/mediawiki/2016/e/eb/T--HUST-China--Experiments-Fig4.png" alt="" class="img-responsive">
 
                 <p class="text-center">Fig4：ideal GFP Fluorescence detection result</p>
 
                 <p class="text-center">Fig4：ideal GFP Fluorescence detection result</p>
Line 359: Line 359:
 
                 <p>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.</p>
 
                 <p>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.</p>
 
                 <h4>Fluorescence detection</h4>
 
                 <h4>Fluorescence detection</h4>
−
                 <p>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. <a href="">see the protein&protein reaction results</a></p>
+
                 <p>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. <a href="">see the results</a></p>
 
                 <img src="https://static.igem.org/mediawiki/2016/f/f9/T--HUST-China--Experiments-Fig6.png" alt="" class="img-responsive">
 
                 <img src="https://static.igem.org/mediawiki/2016/f/f9/T--HUST-China--Experiments-Fig6.png" alt="" class="img-responsive">
 
                 <p class="text-center">Fig6: ideal CIII&Ftsh characterization result</p>
 
                 <p class="text-center">Fig6: ideal CIII&Ftsh characterization result</p>
Line 377: Line 377:
 
                 <p>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.</p>
 
                 <p>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.</p>
 
                 <h4>Fluorescence detection</h4>
 
                 <h4>Fluorescence detection</h4>
−
                 <p>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. <a href="">see the Protein&promoter results</a></p>
+
                 <p>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. <a href="">see the results</a></p>
 
                 <img src="https://static.igem.org/mediawiki/2016/0/06/T--HUST-China--Experiments-Fig10.png" alt="" class="img-responsive">
 
                 <img src="https://static.igem.org/mediawiki/2016/0/06/T--HUST-China--Experiments-Fig10.png" alt="" class="img-responsive">
 
                 <p class="text-center">Fig10: ideal CII&pRE characterization result</p>
 
                 <p class="text-center">Fig10: ideal CII&pRE characterization result</p>
−
                 <p>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.<a href="">see the Protein&promoter results</a></p>
+
                 <p>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.<a href="">see the results</a></p>
 
                 <img src="https://static.igem.org/mediawiki/2016/c/cf/T--HUST-China--Experiments-Fig11.png" alt="" class="img-responsive">
 
                 <img src="https://static.igem.org/mediawiki/2016/c/cf/T--HUST-China--Experiments-Fig11.png" alt="" class="img-responsive">
 
                 <p class="text-center">Fig11: ideal CI&pR characterization result</p>
 
                 <p class="text-center">Fig11: ideal CI&pR characterization result</p>
−
                 <p>Cro and pRM are characterized the same way..<a href="">see the Protein&promoter results</a></p>
+
                 <p>Cro and pRM are characterized the same way..<a href="">see the results</a></p>
 
                 <img src="https://static.igem.org/mediawiki/2016/e/ed/T--HUST-China--Experiments-Fig12.png
 
                 <img src="https://static.igem.org/mediawiki/2016/e/ed/T--HUST-China--Experiments-Fig12.png
 
" alt="" class="img-responsive">
 
" alt="" class="img-responsive">
Line 399: Line 399:
 
                 <h4>Fluorescence detection</h4>
 
                 <h4>Fluorescence detection</h4>
 
                 <p>Microplate spectrophotometer was applied to detect and quantify GFP and RFP expression.</p>
 
                 <p>Microplate spectrophotometer was applied to detect and quantify GFP and RFP expression.</p>
−
                 <p>If successful, it will achieve two stable expression state:GFP expressing state when induced by IAA and RFP by IPTG. <a href="">see Tri-stable function results</a></p>
+
                 <p>If successful, it will achieve two stable expression state:GFP expressing state when induced by IAA and RFP by IPTG. <a href="">see the results</a></p>
 
                 <img src="https://static.igem.org/mediawiki/2016/1/17/T--HUST-China--Experiments-Fig14.png
 
                 <img src="https://static.igem.org/mediawiki/2016/1/17/T--HUST-China--Experiments-Fig14.png
 
" alt="" class="img-responsive">
 
" alt="" class="img-responsive">
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                 <h4>Lactic acid balance function</h4>
 
                 <h4>Lactic acid balance function</h4>
 
                 <p>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.</p>
 
                 <p>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.</p>
−
                <p>  <a href="">see application results</a></p>
 
 
             </article>
 
             </article>
 
         </div>
 
         </div>

Revision as of 21:14, 15 October 2016

Experiments

Experiments

Eukaryote

  • Materials

    more details

    • Strains and vectors:

      more details

      • Pichia pastoris GS115,E.coli DH5α,plasmid pPIC9K

    • Enzymes and reagents:

      more details

      • 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.

    • Media and antibiotics:

      more details

      • 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 the protein expression result

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 the results

Fig4：ideal GFP Fluorescence detection result

Prokaryote

  • Materials

    more details

    • Strains and vectors:

      more details

      • E.coli DH5α,plasmid pSB1C3,plasmid PETDuet-1

    • Enzymes and reagents:

      more details

      • 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

    • Media and antibiotics:

      more details

      • LB medium,ampicillin,chloramphenicol

    • Media and antibiotics:

      more details

      • 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 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 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 results

Fig11: ideal CI&pR characterization result

Cro and pRM are characterized the same way..see the 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 the 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.