July 2
Wet lab
· Digestion of pEGFP-N1 by Ase1 and bamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter
· LB-Chloramphenicol plates (the previous ones were found contaminated)
· Inoculation of pEGFP-N1 for plasmid extraction
· Transformation of pFadBA due chloramphenicol plates contamination

July 3
· Extraction of pEGFP-N1 plasmids by miniprep
· PCR for PPAR-alpha promoter amplification
· Digestion of pEGFP-N1 by Ase1 and bamH1 for PPAR-alpha promoter; , Xba1 and BamH1 for FABP1 promoter

July 4
Wet lab
· Inoculation of BBa_K817002 pFadBA
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· Ran gel for digestion of pEGFP-N1 by Ase1 and bamH1 for PPAR-alpha promoter; Xba1 and BamH1 for FABP1 promoter; Ase1 and Xhol1 for GRP78

July 5
Wet lab
· Extraction of BBa_K817002 (pFadBA)
· Inoculation of pEGFP-N1
· PCR for PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· Gel check, 0.8% gel for previously gDNA extraction
· Ran gel for PCR check of PPAR-alpha promoter and FABP1 promoter
· Poured new LB-Kanamycin plates
· Transformation of BBa_J176171
Dry lab
· Discussion about re-design constructions, possible change for BBa_J176171 as mammalian expression vector
· Primer redesign for PPAR-alpha promoter and FABP1 promoter

July 8
Wet lab
· gDNA extraction from HepG2 cells
· Restriction of BBa_K817002 pFAdBA
· Gel check for gDNA extraction
· Inoculation of BBa_J176171, BBa_K817002 (pFadBA), pEGFP-N1 for plasmid extraction
· New primers for PPAR-alpha promoter and FABP1 promoter arrival
· Ran gel for BBa_K817002 pFadBA restriction check, gel extraction and purification

July 9
Wet lab
· Extraction of BBa_J176171, BBa_K817002 pFadBA and pEGFP-N1 by miniprep
· Restriction of pEGFP-N1 Restriction digestion by Ase1 and bamH1 for PPAR-alpha promoter; Ase1 and Xho1 for GRP78, Xba1 and BamH1 for FABP1 promoter, Pst1 and Not1; followed by gel check, extraction and purification
· Restriction of BBa_J176171 for pFadBA Ase1 and BamH1; FABP1 promoter by Xba1 and Not1
· Ran gel for pEGFP-N1 and BBa_J176171 restriction products
· PCR PPAR-alpha promoter and FABP1 promoter cloning from gDNA
· BBa_J52034 (FADR) transformation

July 10
Wet lab
· Ran gel for PCR check of PPAR-alpha promoter and FAPB1 promoter
· PCR for FABP1 promoter, PCR replication
· BBa_J176171 vector dephosphorylation by antartic phosphatase
· Ligation of BBa_K817002 and pFadBA and BBa_J176171
· BBa_J52034 (FADR) inoculation
· Digestion for BBa_K817002 pFadBA extraction
Gel check for BBa_K817002 pFadBA extraction

July 11
Wet lab
· Gel purification for BBa_K817002 pFadBA extraction
· Gel check for FABP1 promoter PCR product
· PCR clean-up
· BBa_J52034 (FADR) miniprep
· BBa_J52034 restriction by Pst1 HF and Not1 HF
· PCR for PPAR-alpha promoter
Dry lab
Re-design constructions, now using BBa_J176171 as mammalian expression vector for all related constructions

July 12
Wet lab
· Digestion of FABP1 promoter PCR product
· Ran gel for PCR check of PPAR-alpha promoter
· Digest pEGFP-n1 for BBa_J52034 FADR
· Ran gel for pEGFP-n1 for BBa_J52034 FADR followed by gel purification
· BBa_J176171 vector dephosphorylation by Antarctic phosphatase for FABP1 promoter and FADR promoter
Dry Lab
Primers design for pCMV cloning for FADR expression

July 15
Wet lab
· Ligation of FABP1 promoter, eGFP, and BBa_J176171, using 3 pieces ligation
· Transformation of FABP1 promoter, eGFP, and BBa_J176171 ligation
· FABP1 promoter+eGFP +BBa_J176171 ligation restriction check

July 16
Wet Lab
· Miniprep for full construct of FABP1 promoter and pEGFP-N1
· FADR and pEGFP-N1 Backbone parts ligation
· Plasmids extraction for pCMV cloning for FADR
· BBa_K817002 pFadBA promoter extraction by EcoR1 and Pst1 HF
· BBa_J52034 restriction by EcoR1 and Pst1 HF
· Inoculations for full construct of FABP1 promoter and pEGFP-N1
· Streak colonies containing the right construct for FABP1 promoter

July 17
Wet lab
· Plasmid extraction for FABP1 promoter+eGFP+BBa_J176171 by miniprep
· Repeat FadR and pFadBA constructs
· Digest pEGFP-n1 and BBa-J176171 for FADR
· Digestion for BBa_K817002 (pFadBA) extraction
· Gel check and gel extraction for BBa_J176171 for FADR and BBa_K817002 (pFadBA)

July 18
Wet lab
· Gel purification for all digested products
· FABP1 promoter digestion check for whole construct prior transfection
· pFadBA vector de phosphorylation and ligation with eGFP and BBa_J176171
· Transformation of pFadBA+eGFP+BBa_J176171

July 19
Wet lab
· FABP1 promoter preparation for transfection
· Ligation of pFadBA and BBa_J176171 followed by transformation
· PCR for PPAR-alpha promoter cloning
Dry lab
· Design for characterization of the transfected cells with FABP1 construct
· Review for Multiple Sites Mutagenesis
· Ordered primers for pCMV cloning from pEGFP-N1

July 22
Wet lab
· FABP1 construct given for characterization and transfection
· Further colony screening for FABP1 construct, inoculations
· Inoculation of pFadBA and BBa_J176171
· Ran gel for PPAR-alpha promoter PCR products

July 23
Wet lab
· Primers arrival for pCMV cloning
· PCR for pCMV cloning
· Digestion and gel for construction check for FABP1 promoter
· pFadBA gel extraction and purification, followed by vector dephosphorylation BBa_J176171 and ligation, then transformation
· Digestion of pEGFP-N1 for FADR, followed by gel check and extraction

July 24
Wet lab
· DNA purification from gel extraction for digested pEGFP-N1
· Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter
· Ran gel for pCMV cloning from pEGFP-N1
· PCR clean up for pCMV cloning from pEGFP-N1
· PCR for PPAR-alpha promoter with new primers using Taq polymerase
· Transformation of mutagenesis products
· Inoculation of pFadBA ligation colonies

July 25
Wet lab
· Ran gel check PPAR-alpha promoter PCR cloning with new primers using Taq polymerase
· Minprep of pFadBA+eGFP+BBa_J176171 ligation colonies, followed by digestion check

July 26
Wet lab
· Multiple sites mutagenesis for illegal restriction sites for FABP1 promoter
· Ran gel before parental string digestion of mutagenesis products
· Digestion of pEGFP-N1 by Apa1 and Xba1 followed by gel check
· Plasmids preparation according to transfection requirements, construct and controls

July 30
Wet lab
· PPAR-alpha promoter PCR cloning with new primers using Vent polymerase
· Restriction check for pEGFP-N1, using EcoR1, Xba1, Spe1 and Pst1
· BBa_J176171 vector de phosphorylation, followed by ligation with eGFP using T4 Ligase, then transformation into E. Coli strain DH10b
· PCR for PPAR-alpha promoter with new primers using Vent polymerase
Dry lab
· Discussion and re assessment of constructs related to pEGFP-N1

July 31
· Ran gel check for PPAR-alpha promoter PCR cloning with new primers using Vent polymerase
· PCR for pCMV cloning from pEGFP-N1, followed by gel check and PCR clean up
· PCR for PPAR-alpha promoter with reference primers using Vent polymerase
· Ran gel check for PPAR-alpha promoter PCR cloning with reference primers using Vent polymerase

August 2
Wet lab
· Restriction check for EGFP+ BBa_J176171 using EcoR1 and Pst1HF
· Consensus Kozak sequence extraction from BBa_J176171 by EcoR1 and Xba1, followed by gel check and DNA purification
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Vent polymerase
· Genomic DNA extraction from HepG2 cells, Phenol/Chloroform protocol

August 5
Wet lab
· Gel Check for PPAR-alpha promoter PCR promoter cloning, using temperature gradient and every available set of primers designed.
· Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with FADR followed by transformation
· Ligation check of pFadBA+BBa_J176171+eGFP using Age1
· PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase
· gDNA Gel check
Dry lab
· Primers design for BBa_J04450-PSB1C3 Xba1 restriction site removal by site direct mutagenesis

August 6
Wet lab
· Ran gel for PCR for PPAR-alpha promoter, using temperature gradient and every available set of primers designed, using Taq polymerase
· Inoculation of FADR+BBa_J176171

August 7
Wet lab
· Digestion check of BBa_J176171+eGFP by Not1 and Pst1
· Digestion check of BBa_J176171+FADR by Xba1 and HindIII
· Ran gel for digestion check
· Inoculation of FADR+BBa_J176171, colony screening

August 8
Wet lab
· Miniprep for FADR+BBa_J176171, colony screening
· Digestion check of BBa_J176171+FADR by Xba1 and HindIII
· Ran gel for digestion check
· Inoculation of FADR+BBa_J176171, colony screening
· pCMV from cloned from pEGFP-N1adding restriction sites for ligation with eGFP and BBa_J176171, followed by transformation

August 9
Wet lab
· Primers arrival for BBa_J04450-PSB1C3 for site direct mutagenesis
· Digestion check of BBa_J176171+FADR Xba1 and HindIII, Xba1 and Spe1
· Ran gel for digestion check
· Site direct Mutagenesis for BBa_J04450-PSB1C3 removing Xba1 restriction site, followed by gel check before parental string digestion and transformation
· Inoculation of pCMV from cloned from pEGFP-N1adding restriction sites for ligation with eGFP and BBa_J176171

 

August 12
Wet lab
·      Miniprep of pCMV+eGFP+ BBa_J176171, followed by digestion check and gel
·      PCR for pCMV cloning from pEGFP-N1 using Vent polymerase, followed by gel check and PCR clean up
·      Digestion check of BBa_J176171+FADR by Xba1, HindIII and Spe1
·      Ran gel for digestion check
·      pEGFP-N1 digestion for GRP78, extraction pCMV using Ase1 and Xho1, followed by gel check and DNA purification
·      Digestion of BBa_J176171 using single restriction site, Xba1, vector dephosphorylation and ligation with FADR followed by transformation
·      Site direct mutagenesis for BBa_J04450-PSB1C3, followed by gel check before parental digestion with Dpn1 and transformation

 

August 13
Wet lab
·      Primers phosphorylation for FABP1 promoter Multi-site Mutagenesis using T4 PNK
·      Primers phosphorylation for Site Direct Mutagenesis for BBa_J04450-PSB1C3 using T4 PNK
·      Site Direct Mutagenesis for BBa_J04450-PSB1C3 using phosphorylated primers and non phosphorylated primers
·      Overnight culture for Competent cells
·      pDRIVE_hGRP78 arrival, transformation into E. Coli strain DH10b  

 

August 14
Wet lab
·      Nothing done due the typhoon

 

August 15
Wet lab
·      pFadBA+pEGFP-N1 ligation, first vector dephosphorylation using Antarctic phosphatase followed by ligation by T4Ligase and transformation into E. Coli SURE strain.
·      pCMV+eGFP+BBa_J176171 digestion check, followed by gel check
·      Repeat GRP78+pEGFP-N1 ligation, followed by transformation into E. Coli DH10b strain.
·      Digestion check for FADR+BBa_J176171 usign Xba1 and HindIII
·      Inoculations of FADR+ BBa_J176171, pCMV+eGFP+ BBa_J176171, pFadBA+pEGFP-N1, pFadBA+eGFP+ BBa_J176171 followed by gel check

 

August 16
Wet lab
·      Miniprep of FADR+ BBa_J176171, pCMV+eGFP+ BBa_J176171, pFadBA+pEGFP-N1, pFadBA+eGFP+ BBa_J176171
·      Restriction check for FADR+ BBa_J176171 and pFadBA+pEGFP-N1 followed by gel check
·      Inoculations of FADR+ BBa_J176171, pFadBA+pEGFP-N1, pFadBA+eGFP+ BBa_J176171
·      DH105alpha E. Coli competent cells preparation

 

 

August 19
Wet lab
·      Repeat GRP78+pEGFP-N1 ligation, followed by transformation into E. Coli DH10b strain.
·      Miniprep of FADR+ BBa_J176171, pFadBA+pEGFP-N1, pFadBA+eGFP+ BBa_J176171
·      Restriction check for pFadBA+pEGFP-N1 followed by gel check
·      Restriction check for FADR+ BBa_J176171 and pFadBA+pEGFP-N1 followed by gel check
·      Streak colony with the right FADR+BBa_J176171 construct
·      DH5alpha E. Coli competent cells preparation
·       

August 20
Wet lab
·      Digestion check pFadBA+pEGFP-N1 with Pst1, Xba1. Followed by gel check
·      Digestion check for damaged plates of GRP78+pEGFP-N1 using Xba1
·      Digestion check of pFadBA+eGFP+ BBa_J176171 using Age1HF, HindIII and Xba1
·      Site Direct Mutagenesis for illegal restriction sites for FABP1 promoter
·      Inoculate GRP78+pEGFP-N1, pCMV+eGFP+BBa_J176171
·      DH10b E. Coli competent cells preparation

 

August 21
Wet lab
·      Miniprep GRP78+pEGFP-N1, followed by digestion check and gel check using Spe1
·      Miniprep pCMV+eGFP+BBa_J176171 followed by digestion check using Nde1 and Age1HF
·      Digestion check of pFadBA+eGFP+BBa_J176171 using Nde1 , FADR+ BBa_J176171 using Age1
·      Site direct Mutagenesis for FABP1 promoter+eGFP+BBa_J176171
·      PCR for GRP78 promoter cloning from pDRIVE_hGRP78, followed by gel check PCR clean up, digestion using Ase1 and Xho1 and DNA purification

 

August 22
Wet lab
·      DH10b E. Coli competent cells preparation
·      Site direct Mutagenesis for FABP1 promoter+eGFP+BBa_J176171 with phosphorylated primers, followed by gel check before Dpn1 parental string digestion
·      Ligation of GRP78 and pEGFP-N1, first vector dephophorylation and ligation with T4Ligase. Transformed into E. Coli DH10b strain
·      Transformation of pBlueScript KS (+) in to SURE E. Coli
Dry lab
·      Discuss about FABP1 construct vanishing under PCR conditions

 

 

 

August 26
Wet lab
·      FABP1 promoter assessment under PCR conditions
·      BBa_J176171+eGFP assessment under PCR conditions
·      BBa_J176171bassessment under PCR conditions
·      Temperature gradient for FABP1 promoter+BBa_J176171+eGFP construct
·      Inoculate pBlueScript KS (+)

 

August 27
Wet lab
·      DH10b E. Coli competent cells preparation

 

August 28
Wet lab
·      BBa_J176171bassessment under PCR conditions
·      Temperature gradient for FABP1 promoter+BBa_J176171+eGFP construct
·      PCR for FABP1 promoter cloning from gDNA
·      GRP78 ligation to dephosphorylated pEGFP-N1, ligation done using T4 Ligase, transformed into E.Coli DH10b
·      Miniprep pBlueScript KS (+)
·      Digest pBlueScript KS (+) with Xba1 and BamH1HF
·      Restreak BBa_J176171 and inoculate it

 

August 29
Wet lab
·      Miniprep estreaked BBa_J176171
·      GRP78 ligation to dephosphorylated pEGFP-N1, ligation done using T4 Ligase, transformed into E.Coli DH10b
·      Ligation of FABP1 promoter in to pBlueScript KS (+) using T4 Ligase followed by transformation into DH10b E. Coli
·      Transformation of BBa_J176171 into SURE E. Coli

 

August 30
Wet lab
·      Inoculate GRP78+pEGFP-N1
·      Inoculate FABP1 promoter+pBlueScript KS (+)
·      Inoculate BBa_J176171
·      PCR conditions check for BBa_J176171 looking for heat degradation

 

August 31
Wet lab
·      Miniprep of FABP1 promoter+pBlueScript KS (+), BBa_J176171
·      PCR conditions check for BBa_J176171 looking for heat degradation

 

September 1
Wet lab
·      Digestion check for FABP1 promoter+pBlueScript KS (+) using EcoR1. Followed by gel check.

 

September 2
Wet lab
·      Digestion check for FABP1 promoter+pBlueScript KS (+) using EcoR1. Followed by gel check.

 

September 3
Wet lab
·      Restriction check from the damaged plates GRP78+pEGFP-N1 using Xba1
·      Repeat FABP1 promoter and pBlueScript KS (+) ligation, vector Dephosphorylation by Antartic Phosphatase and ligation using T4 Ligase, followed by transformation into DH10b E. Coli

 

September 4
Wet lab
·      GRP78 promoter PCR Cloning from pDRIVE_hGFRP78, followed by PCR cleanup using kit. Then restriction check using Xba1
·      pEGFP-N1 restriction for GRP78 assembly, digested by Ase1 and BamH1, then Gel check, Gel extraction and purification, vector dephosphorilation and ligation by T4 ligase. Transformed into DH10b E. Coli
·      Inoculation of FABP1 promoter+ pBlueScript KS (+)

 

September 5
Wet lab
·      Miniprep of FABP1 promoter+pBlueScript KS (+) then restriction check using Ecor1, followed by gel check.
·      Site Direct Mutagenesis for FABP1 promoter+pBlueScript KS (+), Gel Check

 

September 6
Wet lab
·      Restriction of GRP78 PCR product by Ase1 and Xho1
·      PCR for PPAR-alpha promoter cloning
Dry lab
·      Review on previous Mutagenesis attempts and troubleshooting

 

 

September 9
Wet lab
·      Ran gel for PPAR-alpha promoter cloning
·      pEGFP-N1  digestion for PPAR-alpha promoter and vector dephosphorylation

 

September 10
Wet lab
·      Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check
·      Site Direct Mutagenesis FABP1 promoter+pBlueScript KS(+) then Gel Check

 

September 11
Wet lab
·      Site Direct Mutagenesis FABP1 promoter+pBlueScript KS(+) then Gel Check
·      Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check
·      Digest mutant FABP1 promoter+ pBlueScript KS(+) with Dpn1 then transformed into SURE E. Coli

 

September 12
Wet lab
·      GRP78 PCR product digested by Ase1 and Xho1, ligated with dephosphorylated pEGFP-N1 vector and ligated by T4 ligase, transformed into DH10b E. Coli
·      Inoculations of mutant FABP1 promoter+pBlueScript KS(+)

 

September 13
Wet lab
·      Miniprep of Mutant FABP1 promoter+ pBlueScript KS(+), then restriction check using Ecor1, ran gel
·       Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check

 

September 14
Wet lab
·       Site Direct Mutagenesis for pFRIVE_hGRP78 then Gel Check

 

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