6/22: Received plasmid pFC330, pFC331, pFC332, pFC333 and pFC334 from Technical University of Denmark (Danish: Danmarks Tekniske Universitet)

6/29: Received plasmid pBARGPE1 From Bioresource Collection and Research Center

6/30: Received plasmids pCAMBIA1201 from Academia Sinica

6/30: Received plasmids pCAMBIA1300 from National Chung Hsing University

6/30: Received plasmid pTiB0542 from Academia Sinica

7/4: Received plasmid pVP-EL222 from University of Texas Southwestern

7/4: Received plasmid C120_empty from university of Texas Southwestern

7/4: Received plasmid ARSEF549 from ARSEF of USA

7/9: BBa_E1010 T-streaked onto agar plates

7/10: BBa_E1010 extracted the checked with restriction digestion (EocRI and PstI)

7/11: pBARGPE1 resuspended (from cryovial)

7/11: Transformed pBARGPE1 into competent cells

7/12: pBARGPE1 containing cultures used in 2 in 1

7/12: pBARGPE1 extracted from transformed cultures

7/13: pBARGPE1 checked with restriction digestion (EcoRI和SpeI)

7/13: pC120_empty resuspended (sample from University of Texas Southwestern)

7/13: pC120_empty transformation attempted (failed, no cultures on first plate)

7/13: pVP-EL222 resuspended (sample from University of Texas Southwestern)

7/13: pVP-EL222 transformed into competent cells

7/14: pVP-EL222 did 3 in 1

7/14: pVP-EL222 extracted from transformed cultures and checked with restriction digestion (EcoRI andXhoI)

7/14: pC120_empty transformation

7/15: pC120_empty extracted from transformed cultures and checked with PstI restriction digestion

7/15: pVP-EL222 plasmid stored and transformed cultures cryopreserved

7/16: pC120_empty plasmid stored and transformed cultures cryopreserved

7/16: BBa_E1010 checked with plasmid PCR (Primers: VF2-VR) (failed, no expected product)

7/17: BBa_E1010 checked again with plasmid PCR (failed, probably due to primer VF2-FR malfunction)

7/18: BBa_E1010 checked with plasmid PCR with newly design primers

7/19: BBa_E1010 checked with plasmid PCR with the addition of Mg2+

7/21: pBARGPE1 plasmid stored and transformed cultures cryopreserved

7/22: Received plasmid pAN7-1 from Kaohsiung District Agricultural Research and Extension Station

7/22: Transformed plasmid pAN7-1 into competent cell

7/22: plasmid pAN7-1 did 2 in 1(liquid culture & second plate)

7/22: BBa_E1010 checked with Taq polymerase plasmid PCR (successful)

7/22: pAN7-1 transformation

7/22: pAN7-1 did 2 in 1

7/23: pAN7-1 plasmid extraction

7/23: Extracted plasmid pAN7-1

7/23: Transformed pCAMBIA1300 into competent cells

7/24: pCAMBIA1300 containing cultures used in 2 in 1

7/24: Extracted plasmid pCAMBIA1300

7/24: pCAMBIA1300 digestion check

7/24: pAN7-1 amplified with plasmid PCR (primer: minPgpdA)

7/24: pAN7-1 extracted and transformed cultures cryopreserved

7/24: pAN7-1 did plasmid PCR

7/24: plasmid pAN7-1 stored plasmid and cryopreservation bacterical cultures

7/25: BBa_E1010 amplified with KOD polymerase PCR

7/26: pCAMBIA1300 stored plasmid and transformed cultures cryopreserved

7/26: RFP amplified with KOD polymerase PCR

7/26: RFP digested with EcoRI and BamHI

7/26: Amplified minimal PgpdA promoter on pAN7-1 by doing KOD PCR

7/26: pBARGPE1 digested for pBAR_RFP construction

7/27: PgpdA checked with Taq polymerase plasmid PCR

7/28: Digested minimal PgpdA fragment for ligation with pC120_empty backbone to construct vector pC120_minPgpdA

7/28: pC120_empty ran restriction digestion for ligation with HindIII and BamHI (pC120_empty concentration was too low and contamination of the samples were detected)

7/29: VP-TR amplified with KOD polymerase PCR (failed, contaminated NC)

7/29: pVP-EL222 amplified with plasmid PCR (contaminated NC)

7/29: pBAR_RFP checked with KOD polymerase PCR (contaminated NC)

7/30: pVP-EL222 checked with plasmid PCR with brand new reagents (success, no contamination detected)

7/30: pC120_empty digestion for ligation with HindIII and BamHI

7/30: pBARGPE1 digested (concentration not high enough)

7/31: Transformed C120_minimal PgpdA into competent cells

7/31: C120_minimal PgpdA containing cells used in 2 in 1

7/31: pVP-EL222 amplified with plasmid PCR

7/31: VP-TR trouble shooting

7/31: VP-TR PCR amplification

8/1: pVP-EL222 digested with SpeI and BamHI)

8/1: C120_minimal PgpdA extracted from transformed cells

8/1: pBARGPE1 digested for pBAR_RFP construction

8/1: RFP checked with PCR

8/1: C120_minimal PgpdA checked with restriction digestion

8/2: C120_minimal PgpdA amplified with plasmid PCR

8/2: pBAR_RFP containing cultures used in 3 in 1

8/3: Extracted plasmid pBAR_RFP

8/3: pBAR_RFP did digestion check (wrong)

8/3: C120_minimal PgpdA digested for ligase with RFP

8/3: C120_minimal PgpdA_RFP transformed into competent cell

8/3: PgpdA checked with KOD polymerase plasmid PCR

8/3: PgpdA amplified with KOD polymerase plasmid PCR

8/3: RFP digested with EcoRI and BamHI for ligation

8/4: PgpdA digested for pBAR_PgpdA construction

8/4: C120_minimal PgpdA_RFP extracted from transformed cells

8/4: C120_minimal PgpdA_RFP amplified with plasmid PCR and checked with restriction digestion

8/4: pBARGPE1 digested for pBAR_EL222_TtrpC construction

8/5: pBARGPE1 digested for pBAR_PgpdA construction

8/5: pBAR_PgpdA transformed into competent cell

8/5: Transformed pBAR_RFP into competent cells

8/6: pBAR_RFP did 2 in1

8/6: pBAR_PgpdA containing cultures used in 2 in 1

8/6: C120_minimal PgpdA_RFP plasmid stored and transformed cultures cryopreserved

8/7: pBAR_PgpdA extracted and checked with plasmid PCR and restriction digestion

8/8: Extracted plasmid pBAR_RFP

8/13: pBAR_RFP checked with restriction digestion

8/13: pBAR_RFP checked with plasmid PCR

8/13: mRFP1 PCR amplification

8/13: TtrpC PCR amplification for VP- EL222_RFP

8/14: pBAR_PgpdA plasmid stored and transformed cultures cryopreserved

8/14: C120_minimal PgpdA_RFP digested for ligation with TtrpC

8/15: C120_minimal PgpdA_RFP_TtrpC transformed into competent cell

8/15: pBARGPE1 digested for pBAR_mRFP1_TtrpC construction

8/15: pVP-EL222 amplified with PCR

8/15: TtrpC and pVP- EL222 PCR amplificaiton

8/16: TtrpC digestion

8/16: mRFP1 digestion

8/16: pBARGPE1 digested for pBAR_EL222_TtrpC construction

8/16: pBAR_EL222_TtrpC transformed into competent cell

8/16: pBAR_EL222_TtrpC containing cultures used 2 in 1

8/17: pBAR_EL222_TtrpC extracted and attempted to amplify with plasmid PCR------we did not amplify the target sequence (Hence, we tried ligation again)

8/17: C120_minimal PgpdA_RFP_TtrpC extracted and checked with restriction digestion

8/17: C120_minimal PgpdA_RFP_TtrpC amplified with plasmid PCR

8/17: pBAR _RFP_TtrpC transformed into competent cell

8/17: pBAR _RFP_TtrpC containing cultures used in 2 in 1

8/18: pBAR _RFP_TtrpCdid extracted from transformed cultures and checked with restriction digestion

8/18: C120_minimal PgpdA_RFP_TtrpC plasmid stored and transformed bacterial cultures cryopreserved

8/18: pBAR_PgpdA digested for ligation with pVP-EL222 and TtrpC

8/18: pBAR_EL222_TtrpC transformed into competent cell

8/18: pBAR_EL222_TtrpC containing cultures used in 3 in 1

8/18: Amplified MCL1 promoter (PMCL1) from genome (Taq PCR from Metarhizium anisopliae ARSEF549 genomic DNA)

8/19: pBAR _RFP_TtrpC checked with restriction digestion------the incorrect bands appeared in gel electrophoresis results

8/19: mRFP1 PCR amplification (failed)

8/19: pBAR_EL222_TtrpC extracted from transformed cultures, checked with restriction enzyme digestion and plasmid PCR (failure)

8/19: pBAR_PgpdA_EL222_TtrpC transformed into competent cell

8/20: pVP-EL222 amplified with PCR than digested

8/21: pBAR_PgpdA_EL222_TtrpC extracted and checked with restriction digestion (failure)

8/21: mRFP1 PCR amplification

8/21: mRFP1 digestion

8/21: pBAR_PgpdA_EL222_TtrpC transformed into competent cell

8/22: PMCL1 Taq polymerase PCR amplification test run (failed, switched program afterwards)

8/23: PMCL1 KOD polymerase PCR amplification test run

8/23: PMCL1 Taq polymerase PCR amplification test run (with adjusted Mg2+ concentrations)

8/23: TtrpC PCR amplification

8/23: TtrpC digestion

8/23: pBAR_PgpdA_EL222_TtrpC containing cultures used in 3 in 1

8/23: BBa_E0040 containing cells T-streaked onto agar plates (failed to grow due to using incorrect antibiotics

8/24: pBAR_PgpdA_EL222_TtrpC checked with restriction digestion

8/24: pBAR_PgpdA_EL222_TtrpC amplified with plasmid PCR

8/24: PMCL1 genome PCR amplification test run

8/24: NahR_GFP resuspended (from 2015 NYMU iGem team’s cryopreservation bacterical cultures)

8/24: NahR_GFP containing cultures used in 2 in 1

8/25: PMCL1 PCR amplification test run

8/25: pVP-EL222 checked separately with plasmid PCR with redesigned primers

8/25: mRFP1 checked with PCR due to multiple failed double ligation

8/26: mRFP1 checked with KOD polymerase PCR

8/26: mRFP1 amplified with KOD polymerase PCR

8/26: pVP-EL222 checked with plasmid PCR due to multiple failed ligations

8/26: pVP-EL222 checked with restriction digestion due to multiple failed ligations

8/26: pVP-EL222 checked with Taq polymerase plasmid PCR with remixed primers

8/26: PMCL1 KOD polymerase PCR amplification test run

8/26: TtrpC KOD PCR check(with remixed pVP- EL222 primers)

8/27: TtrpC KOD PCR amplification

8/27: BBa_E0040 containing cells T-streaked onto agar plates

8/27: mRFP1 and TtrpC PCR ligation

8/27: pBARGPE1 checked with restriction digestion due multiple failed attempts to ligate pBARGPE1 with various inserts

8/27: pVP-EL222 checked with KOD polymerase plasmid PCR with remixed primers

8/27: pVP-EL222 amplified with PCR (use for ligation with both pBAR_PgpdA and PMCL1)

8/28: RFP_TtrpC digested for ligation with pBARGPE1------ the concentration was not high enough

8/28: RFP and TtrpC did PCR to ligase(failure, we change the volume of the primer and Mg2+)

8/28: EL222 and TtrpC ran ligation PCR ------- NC had band

8/28: EL222 and TtrpC ran ligation PCR ------ with remixed primer

8/28: pVP-EL222 and TtrpC PCR ligation ------- NC had bands

8/28: pVP-EL222 and TtrpC PCR ligation ------- used remixed primer

8/29: pVP-EL222 and TtrpC PCR ligation ------- the concentration was not high enough after gel extraction

8/29: EL222 and TtrpC ran ligation PCR ------ the concentration was not high enough after gel extraction

8/31: EL222 and TtrpC ran ligation PCR

8/31: pBAR_RFP digestion(to transformed into meta)

8/31: RFP_TtrpC digested for ligase with pBARGPE1 (the band existed on wrong site)

8/31: pVP-EL222 and TtrpC PCR ligation

8/31: PMCL1 Taq polymerase PCR amplification

9/1: PMCL1 PCR amplification test run

9/1: PMCL1 KOD polymerase PCR amplification

9/1: PMCL1 polymerase PCR amplification

9/1: RFP_TtrpC digested

9/1: pVP-EL222 and TtrpC PCR ligase

9/1: EL222 and TtrpC ran ligation PCR

9/2: EL222-TtrpC digestion

9/2: pBARGPE1 digested for pBAR_EL222_TtrpC construction

9/2: pBAR _RFP_TtrpC transformed into competent cell

9/2: pBAR_PgpdA digested for ligation with EL222_TtrpC

9/2: PMCL1 KOD polymerase PCR amplification

9/2: PMCL1 KOD polymerase PCR amplification

9/3: PMCL1 KOD polymerase PCR amplification

9/3: pBAR_EL222_TtrpC transformed into competent cell

9/3: pBAR_EL222_TtrpC containing cultures used in 3 in 1

9/3: pBAR_PgpdA digested for ligation with EL222_TtrpC

9/3: pBAR_PgpdA_EL222_TtrpC transformed into competent cell

9/4: pBAR_PgpdA_EL222_TtrpC containing cultures used in 3 in 1

9/4: pBAR_PgpdA_EL222_TtrpC extracted from transformed cultures

9/4: pBAR _RFP_TtrpC did plasmid PCR

9/4: PMCL1 KOD polymerase PCR amplification

9/5: pBAR _RFP_TtrpC transformed into competent cell ------ there was no E.coli on the first plate

9/5: mRFP1 amplified with KOD polymerase PCR

9/5: pBAR_PgpdA_EL222_TtrpC amplified with plasmid PCR

9/5: pBAR_EL222_TtrpC checked with restriction enzyme digestion

9/6: EL222-TtrpC digestion

9/6: pBAR_EL222_TtrpC checked with restriction enzyme digestion

9/6: pBAR_PgpdA_EL222_TtrpC checked with restriction digestion (failure)

9/6: pBAR_PgpdA_EL222_TtrpC checked with restriction digestion (we changed enzymes)

9/6: EL222-TtrpC checked with restriction digestion

9/6: EL222-TtrpC digested with restriction enzyme

9/6: BBa_E0040 streaked onto agar plates

9/6: BBa_K118400 resuspended from 2016 iGEM Kit plate 4

9/6: BBa_K118400 transformed into competent cells

9/6: pBAR _RFP_TtrpC transformed into competent cell

9/6: PMCL1 KOD polymerase PCR amplification

9/6: PMCL1 KOD polymerase PCR amplification

9/7: PMCL1 KOD polymerase PCR amplification

9/7: pBAR _RFP_TtrpC transformed into competent cell

9/7: pBAR _RFP_TtrpC did 2 in 1

9/7: pBAR _RFP_TtrpC did 3 in 1

9/7: BBa_E0040 cultures used in 2 in 1

9/7: pBAR_EL222 PCR

9/7: BBa_K118400 containing culture used in 2 in 1

9/7: pVP-EL222 amplified with PCR (use for ligation with both pBAR_PgpdA and PMCL1)

9/8: pVP-EL222 amplified with PCR (use for ligation with both pBAR_PgpdA and PMCL1)

9/8: BBa_K118400 extraction extracted from transformed cultures

9/8: BBa_K118400 amplified with plasmid PCR

9/8: BBa_E0040 extracted from transformed culture

9/8: pBAR _RFP_TtrpC plasmid PCR

9/8: pBAR _RFP_TtrpC digestion check

9/8: pBAR _RFP_TtrpC digestion check

9/8: pBAR _RFP_TtrpC did plasmid extraction

9/8: PMCL1 KOD polymerase PCR amplification

9/9: PMCL1 KOD polymerase PCR amplification

9/9: pBAR _RFP_TtrpC did digestion check

9/9: BBa_E0040 amplified in plasmid PCR

9/9: mRFP1amplified with KOD polymerase PCR

9/9: BBa_E0040 checked with restriction digestion (successful)

9/9: BBa_K118400 checked with restriction digestion (the gel had faulty matrix)

9/9: BBa_K118400 checked with restriction digestion (confirmed, successful)

9/9: BBa_K1184000 use KOD polymerase to amplify the target part KillerRed (try)

9/9: pBAR_PgpdA_EL222_TtrpC checked with restriction digestion

9/9: pBAR_PgpdA_EL222_TtrpC transformed into competent cell( there was no E.coli on our first plate )

9/9: pBARGPE1 digested for pBAR_mRFP1_TtrpC construction

9/10: pBARGPE1 digested for pBAR_PgpdA_EL222_TtrpC construction

9/10: pBAR_PgpdA_EL222_TtrpC transformed into competent cell

9/10: BBa_K1184000 use KOD polymerase to amplify the target part KillerRed (really amplified)

9/10: RFP and TtrpC use primer to ligase

9/10: RFP_TtrpC PCR to amplify the target sequence

9/10: Digested KillerRed fragment and its backbone pBARGPE1 with BamHI and EcoRI for ligation to construct the vector pBAR_KR

9/10: Digested KillerRed fragment and its backbone pBARGPE1 with BamHI and EcoRI again

9/10: Amplified PMCL1 from genome (KOD polymerase)

9/10: Amplified PMCL1 from genome (KOD polymerase)

9/10: Standard part PgpdA amplification PCR with Taq polymerase

9/11: Standard part PgpdA amplification PCR with KOD polymerase

9/11: pBARGPE1 digested for pBAR_PgpdA_EL222_TtrpC construction

9/11: TtrpC KOD PCR amplification

9/11: TtrpC KOD PCR amplification

9/11: pBAR_PgpdA_EL222_TtrpC containing culture used in 3 in 1

9/11: pBAR_KR transformed into competent cell

9/11: pBAR_KR transformed into competent cell

9/12: pBAR_KR performed plasmid extraction and plasmid PCR (successful)

9/12: pBAR_PgpdA_EL222_TtrpC checked with restriction digestion

9/12: pBAR_PgpdA_EL222_TtrpC amplified with plasmid PCR

9/12: Successfully constructed the plasmid pBAR_PgpdA_EL222_TtrpC

9/12: Standard part PgpdA mutation PCR (mutated PstI)

9/12: Standard part PMCL1 amplification PCR with KOD polymerase

9/13: Standard Part PMCL1 mutation PCR (mutated SpeI)

9/13: Standard part TtrpC amplification PCR with KOD polymerase

9/13: pBAR _RFP_TtrpC transformed into competent cell

9/13: RFP_TtrpC PCR to amplify the target sequence

9/13: pBAR _RFP_TtrpC did 3 in 1

9/13: pBAR _RFP_TtrpC digestion check

9/14: pBAR _RFP_TtrpC plasmid extraction

9/14: RFP_TtrpC digested

9/14: K118400 and TtrpC used PCR to amplify the target sequence

9/14: KR_TtrpC used PCR to amplify the target sequence

9/15: KR_TtrpC digested with restriction enzymes

9/15: Standard part TtrpC mutation PCR (mutated XbaI)

9/15: pBARGPE1 digested for pBAR_KR_TtrpC construction

9/16: pBAR_KR_TtrpC transformed into competent cell

9/16: Transformed pBAR_KR_TtrpC into competent cell

9/16: Digested pBAR_KR for transforming into Metarhizium anisopliae

9/16: Extracted plasmid BBa_K118400

9/16: pBAR_EL222_TtrpC did plasmid PCR

9/16: pBAR_EL222_TtrpC did plasmid PCR (confirmed successfully)

9/16: pBAR _RFP_TtrpC did digestion check

9/17: pBAR_RFP_TtrpC did digestion check

9/17: pBAR_EL222_TtrpC plasmid stored and transformed cultures cryopreserved

9/17: pBAR_KR_TtrpC did 3 in 1

9/17: Extracted plasmid pBAR_KR_TtrpC

9/18: Transformed pBAR_RFP_TtrpC into competent cell

9/18: pBAR_RFP_TtrpC did 3 in 1

9/18: pBAR_KR_TtrpC did plasmid PCR and digestion check

9/18: BBa_E0040 did plasmid PCR

9/19: Amplified GFP from plasmid BBa_E0040

9/19: Extracted plasmid BBa_E0040

9/20: KR_TtrpC digestion

9/20: Transformed pBAR_KR_TtrpC into competent cell

9/20: pBAR_KR_TtrpC did 3 in 1

9/20: Extracted plasmid pBAR_KR_TtrpC

9/20: pBAR_KR_TtrpC did digestion check

9/20: Extracted plasmid pBAR_RFP_TtrpC

9/20: pBAR_RFP_TtrpC did digestion check

9/20: RFP_TtrpC digestion

9/21: pBAR_KR_TtrpC did digestion check

9/21: pBAR_RFP_TtrpC did digestion check

9/21: pBAR_RFP_TtrpC did 2 in 1

9/21: pBARGPE1 and RFP_TtrpC digestion

9/22: Amplified RFP_TtrpC with KOD polymerase

9/22: Extracted plasmid pBAR_KR_TtrpC

9/22: pBAR_KR_TtrpC did plasmid PCR

9/22: pMCL1 and pBAR_KR_TtrpC digestion (pMCL1 was not digested successfully)

9/22: pBAR_PgpdA_EL222_TtrpC digested for transforming into Metarhizium anisopliae

9/23: Amplified pMCL1 with KOD polymerase

9/24: pBAR_RFP_TtrpC did digestion check

9/24: Extracted plasmid pBAR_RFP_TtrpC (from 9/5 plate)

9/25: Transformed pBAR_RFP_TtrpC into competent cell

9/25: RFP_TtrpC digestion

9/25: Amplified RFP-TtrpC with KOD polymerase

9/25: Amplified RFP_TtrpC with KOD polymerase

9/25: pMCL1 and pBAR_KR_TtrpC digestion

9/25: Transformed pBAR_pMCL1_KR_TtrpC into competent cell

9/25: Transformed pBAR_pMCL1_KR_TtrpC into competent cell

9/25: pBAR_pMCL1_KR_TtrpC did 3 in 1

9/26: Extracted plasmid pBAR_pMCL1_KR_TtrpC

9/26: pBAR_RFP_TtrpC did 2 in 1

9/26: Extracted plasmid pBAR_RFP_TtrpC

9/26: pBAR_RFP_TtrpC did digestion check

9/26: pBAR_RFP_TtrpC did digestion check

9/26: Amplified pMCL1 with KOD polymerase

9/27: Amplified pMCL1 with KOD polymerase

9/28: pBAR_pMCL1_KR_TtrpC did digestion check

9/28: pBAR_RFP_TtrpC did plasmid PCR

9/28: Successfully constructed the plasmid pBAR_RFP_TtrpC

9/29: Amplified pMCL1 with KOD polymerase

9/30: pBAR_pMCL1_KR_TtrpC did digestion check

9/30: Received the junction primer of GFP-TtrpC

9/30: GFP_TtrpC ran ligation PCR

10/1: GFP_TtrpC ran ligation PCR

10/2: GFP_TtrpC digested for ligation with pBARGPE1

10/2: Transformed pBAR_GFP_TtrpC into competent cells

10/2: pBARGPE1 digested for pBAR_GFP_TtrpC construction

10/2: pBARGPE1 resuspended (from cryopreservation)

10/3: Extracted plasmid pBARGPE1

10/3: pBAR_GFP_TtrpC containing cultures used in 2 in 1

10/3: Extracted plasmid pBAR_GFP_TtrpC

10/3: pBAR_GFP_TtrpC digestion check(failure)

10/3: pBAR_GFP_TtrpC PCR check(failure)

10/3: Transformed pBAR_GFP_TtrpC into competent cells

10/3: pBARGPE1 checked with restriction digestion (correct)

10/4: pBARGPE1 plasmid stored

10/4: pBAR_GFP_TtrpC containing cultures used in 2 in 1

10/4: pBAR_GFP_TtrpC plasmid extraction

10/4: pBAR_GFP_TtrpC digestion check