Team:NEFU China/Protocol

Notebook-2018

Protocol

Cultural Conditions

KEY： Saccharomyces cerevisiae YPH499


  1. Transformation culture conditions： incubator of 30℃， plates with SD/-Leu-agar solid medium.
  2. Inoculation culture conditions: a 30℃ constant temperature shaker with 220 rpm in SD/-Leu liquid medium.

Lock and Information: Saccharomyces cerevisiae YPH499


  1. Transformation culture conditions: incubator of 30℃， plates with SD/-Ura-agar solid medium.
  2. Inoculation culture conditions: a 30℃ constant temperature shaker with 220 rpm in SD/-Ura liquid medium.

pFig2c-Bax: Saccharomyces cerevisiae YPH499


  1. Transformation culture conditions： incubator of 30℃， plates with SD/-Ura-agar solid medium.
  2. Inoculation culture conditions: a 30℃ constant temperature shaker with 220 rpm in SD/-Ura liquid medium.
  3. Inducible expression conditions: a 30℃ constant temperation shaker with 220 rpm in SD/-Ura liquid medium contaning different concentrations of alpha factor.

Vector Construction

Vector construction of Lock and Information：


  1. CYC promoter, CYC1 terminator were amplified from genomic DNA of Saccharomyces cerevisiae YPH499 with corresponding primers respectively. And the DNA of the lock and information fragment was synthesized by company with restriction enzyme cutting sites HindIII, EcoRI and EcoRI, NheI.
  2. Modified CYC1 promoter and CYC1 terminator by adding the HA1 or HA2 by using PCR.
  3. Four fragments of HA1-CYC promoter, Stem loop (the lock), Information and Cyc1 terminator-HA2 the were double-digested with SacI-HindIII, HindIII-EcoRI, EcoRI-NheI, and NheI-BamHI, respectively.
  4. Ligated them into an expression vector pesc-Ura by T4 ligase.
  5. To detemine whether the vector is constructed successfully, conoly PCR and sequencing were done.

Vector construction of Lock and EGFP：


  1. CYC promoter, CYC1 terminator were amplified from genomic DNA of Saccharomyces cerevisiae YPH499 with corresponding primers respectively. And the DNA of the lock was synthesized by company with restriction enzyme cutting sites HindIII, EcoRI. The EGFP gene was amplified from palsmid pEGFP-N2, and modified it by adding EcoRI and NheI respectively.
  2. Modified CYC1 promoter and CYC1 terminator by adding the HA1 or HA2 by using PCR.
  3. Four fragments of HA1-CYC promoter, Stem loop (the lock), EGFP and Cyc1 terminator-HA2 the were double-digested with SacI-HindIII, HindIII-EcoRI, EcoRI-NheI, and NheI-BamHI, respectively.
  4. Ligated them into an expression vector pesc-Ura by T4 ligase.
  5. To detemine whether the vector is constructed successfully, conoly PCR and sequencing were done.

Vector construction of Lock and Gluc：


  1. CYC promoter, CYC1 terminator were amplified from genomic DNA of Saccharomyces cerevisiae YPH499 with corresponding primers respectively. And the DNA of the lock was synthesized by company with restriction enzyme cutting sites HindIII， EcoRI. The Gluc gene was amplified from palsmid pGluc-Basic2, and modified it by adding EcoRI and NheI respectively.
  2. Modified CYC1 promoter and CYC1 terminator by adding the HA1 or HA2 by using PCR.
  3. Four fragments of HA1-CYC promoter, Stem loop (the lock), Gluc and Cyc1 terminator-HA2 the were double-digested with SacI-HindIII, HindIII-EcoRI, EcoRI-NheI, and NheI-BamHI, respectively.
  4. Ligated them into an expression vector pesc-Ura by T4 ligase.
  5. To detemine whether the vector is constructed successfully, conoly PCR and sequencing were done.

Vector construction of Key:


  1. RPR1 promoter was amplified from plasmid named pRPR1_gRNA_handle_RPR1t.
  2. Modified RPR1 promoter by adding the Key sequence and SpeI, XhoI by using PCR.
  3. The fragment of pRPR1-Key was double-digested with SpeI and XhoI respectively.
  4. Ligated it into an expression vector pRPR1_gRNA_handle_RPR1t by T4 ligase.
  5. To detemine whether the vector is constructed successfully, conoly PCR and sequencing were done.

Vector construction of suicide switch:


  1. Bax (alpha) Gene was amplified from genomic DNA of 293T cell with corresponding primers respectively.
    Fig2C promoter fragment was from iGEM parts registry.
  2. Modified Bax fragment by adding SpeI and HindIII by using PCR.
  3. Two fragments of Fig2C promoter, Bax gene were double-digested with EcoRI-SpeI, and SpeI-HindIII, respectively.
  4. Ligated the fragments into an expression vector pesc-Ura by T4 ligase.
  5. To detemine whether the vector is constructed successfully, conoly PCR and sequencing were done.

 

Vector construction of pFig2C-EGFP


  1. Fig2C promoter fragment was from iGEM parts registry. The EGFP gene was amplified from palsmid pEGFP-N2, and modified it by adding SpeI and HindIII respectively.
  2. Two fragments of Fig2C promoter, EGFP gene were double-digested with EcoRI-SpeI, and SpeI-HindIII, respectively.
  3. Ligated the fragments into an expression vector pesc-Ura by T4 ligase.
  4. To detemine whether the vector is constructed successfully, conoly PCR and sequencing were done.

Lock and key interaction verification

Qualitative verification using EGFP


  1. Transform expression plasmid pRPR1-Key and pCYC-Lock-EGFP
    into YPH499. Plate on SD/-Leu-Ura selection plates and incubate at 30℃ for 36 hours.
  2. Resupend a single colony in 10ml SD/-Ura liquid medium
  3. Shake at 30℃ to OD600nm = 0.6
  4. 500ul of the bacterial solution was taken out, a picture was prepared, and the expression of EGFP was observed with a fluorescence microscope.

Quantitative verification using Gluc


  1. Transform expression plasmid pRPR1-Key and pCYC-Lock-Gluc into YPH499. Plate on SD/-Leu-Ura selection plates and incubate at 30℃ for 36 hours.
  2. Resupend a single colony in 10ml SD/-Ura liquid medium
  3. Shake at 30℃ to OD600nm = 0.6
  4. 500ul of bacterial solution was taken for ultrasonic cell disruption, and luciferase substrate Coelenterazine was added to detect the expression of luciferase Gluc.

Fig2C promoter verification of α-factor response

Qualitative verification


  1. Transform expression plasmid pFig2C-EGFP into YPH499. Plate on SD/-Leu-Ura selection plates and incubate at 30℃ for 36 hours.
  2. Resupend a single colony in 10ml SD/-Ura liquid medium
  3. Shake at 30℃ to OD600nm = 0.6
  4. 500ul of the bacterial solution was taken out, a picture was prepared, and the expression of EGFP was observed with a fluorescence microscope.

Quantitative verification by q-PCR


  1. Transform expression plasmid pFig2C-EGFP into YPH499. Plate on SD/-Leu-Ura selection plates and incubate at 30℃ for 36 hours.
  2. Resupend a single colony in 10ml SD/-Ura liquid medium
  3. Shake at 30℃ to OD600nm = 0.6
  4. Take 1ml of bacterial solution to extract RNA, perform reverse transcription.
  5. Perform quantitative PCR using reverse transcription products as templates.

Verification of homologous recombination

Qualitative verification using EGFP


  1. The constructed plasmid pCYC-Lock-EGFP was subjected to PCR to obtain a HA1-pCYC-Lock-EGFP-tCYC-HA2 fragment. 10ul of PCR product was taken, purified and transformed. Plate on SD/-Leu-Ura selection plates and incubate at 30℃ for 36 hours.
  2. Resupend a single colony in 10ml SD/-Ura liquid medium
  3. Shake at 30℃ to OD600nm = 0.6
  4. 500ul of the bacterial solution was taken out, a picture was prepared, and the expression of EGFP was observed with a fluorescence microscope.

PCR verification


  1. The constructed plasmid pCYC-Lock-EGFP was subjected to PCR to obtain a HA1-pCYC-Lock-EGFP-tCYC-HA2 fragment. 10ul of PCR product was taken, purified and transformed. Plate on SD/-Leu-Ura selection plates and incubate at 30℃ for 36 hours.
  2. Resupend a single colony in 10ml SD/-Ura liquid medium
  3. Shake at 30℃ to OD600nm = 0.6
  4. 1 ml of the bacterial solution was taken for DNA extraction.
  5. The obtained genomic DNA was subjected to PCR using a specific primer to verify whether the homologous recombination was successful.
 

Information expression verification

  1. The constructed plasmid pCYC-Lock-EGFP was subjected to PCR to obtain a HA1-pCYC-Lock-EGFP-tCYC-HA2 fragment. 10ul of PCR product was taken, purified and transformed. Plate on SD/-Leu-Ura selection plates and incubate at 30℃ for 36 hours.
  2. Resupend a single colony in 10ml SD/-Ura liquid medium
  3. Shake at 30℃ to OD600nm = 0.6
  4. 1 ml of the bacterial solution was taken for DNA extraction.
  5. The obtained genomic DNA was subjected to PCR using a specific primer to verify whether the homologous recombination was successful.
  6. Take 1ml of bacterial solution to extract RNA, perform reverse transcription to obtain the cDNA.
  7. The cDNA was subjected to PCR using specific primers, and the product was purified and submitted to the company for sequencing.