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

Line 423: Line 423:
 
                 <p>Three pairs of proteins and promoters were examined to confirm the property of their interactions.</p>
 
                 <p>Three pairs of proteins and promoters were examined to confirm the property of their interactions.</p>
 
                 <h4>PCR and Plasmid Construction:</h4>
 
                 <h4>PCR and Plasmid Construction:</h4>
−
                 <p><span style="text-weight:bold">CII and pRE:</span> 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.</p>
+
                 <p><span style="text-weight:bold">CII and pRE:</span> 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.</p>
 
                 <img src="https://static.igem.org/mediawiki/2016/e/ee/T--HUST-China--Experiments-Fig7.png" alt="" class="img-responsive">
 
                 <img src="https://static.igem.org/mediawiki/2016/e/ee/T--HUST-China--Experiments-Fig7.png" alt="" class="img-responsive">
 
                 <p class="text-center">Fig7: CII&pRE interaction characterization plasmid and its control</p>
 
                 <p class="text-center">Fig7: CII&pRE interaction characterization plasmid and its control</p>
−
                 <p><span style="text-weight:bold">CI and pR:</span> 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.</p>
+
<style type="text/css">
 +
table.tableizer-table {
 +
font-size: 14px;
 +
border: 1px solid #CCC;
 +
font-family: Arial, Helvetica, sans-serif;
 +
}
 +
.tableizer-table td {
 +
padding: 4px;
 +
margin: 3px;
 +
border: 1px solid #CCC;
 +
}
 +
.tableizer-table th {
 +
background-color: #767474;
 +
color: #FFF;
 +
font-weight: bold;
 +
}
 +
</style>
 +
<table class="tableizer-table">
 +
<thead><tr class="tableizer-firstrow"><th>Part Number</th><th>Description</th></tr></thead><tbody>
 +
<tr><td>BBa_K2036032</td><td>CII-TT-pRE-RBS-GFP-LVAssrAtag</td></tr>
 +
<tr><td>BBa_K2036011</td><td>pRE-GFP-LVAssrAtag</td></tr>
 +
</tbody></table>
 +
                 <p><span style="text-weight:bold">CI and pR:</span> 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.</p>
 
                 <img src="https://static.igem.org/mediawiki/2016/8/8f/T--HUST-China--Experiments-Fig8.png" alt="" class="img-responsive">
 
                 <img src="https://static.igem.org/mediawiki/2016/8/8f/T--HUST-China--Experiments-Fig8.png" alt="" class="img-responsive">
−
                 <p class="text-center">Fig8:CI&pR interaction characterization plasmid and its control</p>
+
                 <p class="text-center">Fig8: CI&pR interaction characterization plasmid and its control</p>
−
                 <p><span style="text-weight:bold">CI and pR:</span>The same as above.</p>
+
<style type="text/css">
 +
table.tableizer-table {
 +
font-size: 14px;
 +
border: 1px solid #CCC;
 +
font-family: Arial, Helvetica, sans-serif;
 +
}
 +
.tableizer-table td {
 +
padding: 4px;
 +
margin: 3px;
 +
border: 1px solid #CCC;
 +
}
 +
.tableizer-table th {
 +
background-color: #797475;
 +
color: #FFF;
 +
font-weight: bold;
 +
}
 +
</style>
 +
<table class="tableizer-table">
 +
<thead><tr class="tableizer-firstrow"><th>Part Number</th><th>Description</th></tr></thead><tbody>
 +
<tr><td>BBa_K2036016</td><td>pR-GFP-LVAssrAtag</td></tr>
 +
<tr><td>BBa_K2036017</td><td>CI-TT-pR-RBS-GFP-LVAssrAtag</td></tr>
 +
</tbody></table>
 +
                 <p><span style="text-weight:bold">Cro and pRM:</span>The same method as above.</p>
 
                 <img src="https://static.igem.org/mediawiki/2016/a/a1/T--HUST-China--Experiments-Fig9.png" alt="" class="img-responsive">
 
                 <img src="https://static.igem.org/mediawiki/2016/a/a1/T--HUST-China--Experiments-Fig9.png" alt="" class="img-responsive">
−
                 <p class="text-center">Fig9:Cro&pRM interaction characterization plasmid and its control</p>
+
                 <p class="text-center">Fig9: Cro&pRM interaction characterization plasmid and its control</p>
−
                 <h4>Transformation and induction</h4>
+
<style type="text/css">
−
                 <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>
+
table.tableizer-table {
 +
font-size: 14px;
 +
border: 1px solid #CCC;
 +
font-family: Arial, Helvetica, sans-serif;
 +
}
 +
.tableizer-table td {
 +
padding: 4px;
 +
margin: 3px;
 +
border: 1px solid #CCC;
 +
}
 +
.tableizer-table th {
 +
background-color: #8B8788;
 +
color: #FFF;
 +
font-weight: bold;
 +
}
 +
</style>
 +
<table class="tableizer-table">
 +
<thead><tr class="tableizer-firstrow"><th>Part Number</th><th>Description</th></tr></thead><tbody>
 +
<tr><td>BBa_K2036009</td><td>pRM-GFP-LVAssrAtag</td></tr>
 +
<tr><td>BBa_K2036010</td><td>Cro-TT-pRM-RBS-GFP-LVAssrAtag</td></tr>
 +
</tbody></table>
 +
                 <h4>Transformation and Induction:</h4>
 +
                 <p>All plasmids and their control groups were transformed into E.coli DH5α cells by standard competent cell transformation method. (See details in Protocol) Then they were grown in LB medium containing 1‰ ampicillin. IPTG acts as the inducer to activate T7 promoter in the 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. </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. </p>

Revision as of 12:11, 18 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（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）.

    • Media and Antibiotics:

      more details

      • LB medium（）, YPD medium（）, Ampicillin（Sangon A100741-0025）

    • Equipment:

      more details

      • 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.

Fig1: 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. (See details in Protocol) 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.

Fig2: Eukaryotic bi-stable function characterized plasmid assembly work flow

Part NumberDescription
BBa_K2036030PP2CA-TTADH1-pCyc-Kozak-Snrk2.2-TTADH1-pCyc-Kozak-ABF2-TTADH1-prd29A-Kozak-GFP-LVAssrAtag

Fig3: 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.

Fig4：Proposed 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（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）

    • Media and Antibiotics:

      more details

      • LB medium（）, Ampicillin（Sangon A100741-0025）, Chloramphenicol（Sangon A100230-0025）

    • Equipment:

      more details

      • 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.

Fig5: Protein and protein interaction characterized plasmid assembly work flow

Part NumberDescription
BBa_K2036014RBS-CIII-RBS-CIII-RBS-CII-TT-pRE-RBS-GFP-LVAssrAtag
BBa_K2036015RBS-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. (See details in Protocol) 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.

Fig6: 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.

Fig7: CII&pRE interaction characterization plasmid and its control

Part NumberDescription
BBa_K2036032CII-TT-pRE-RBS-GFP-LVAssrAtag
BBa_K2036011pRE-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.

Fig8: CI&pR interaction characterization plasmid and its control

Part NumberDescription
BBa_K2036016pR-GFP-LVAssrAtag
BBa_K2036017CI-TT-pR-RBS-GFP-LVAssrAtag

Cro and pRM:The same method as above.

Fig9: Cro&pRM interaction characterization plasmid and its control

Part NumberDescription
BBa_K2036009pRM-GFP-LVAssrAtag
BBa_K2036010Cro-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. (See details in Protocol) 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. 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.

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.

Fig11: ideal CI&pR characterization result

Cro and pRM are characterized the same way.

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.

Fig14: ideal tri-stable characterization result


Application circuit construction

Materials: The same as Prokaryote’s

lactic acid balance 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.