Week 1
23-06-01 Miniprep Pcargo and Transformation of Entry Vectors
23-06-02 PCR Amplification and Diagnostic Age of Pz Promoter
23-06-03 Pz Fragment Purification Glycerol Stocks of Entry Vectors and Pz Miniprep of Genes Entry Vectors and Gga Reactions
23-06-04 Transformation of Assembly Vectors with Drop Dialysis
Week 2
23-06-05 Re Gga of Initial Assemblies
23-06-06 Transformation of Re Gga for Assembly Vectors Plating of Equol Strains Inoculation of Eql Donor Strain
23-06-07 Plating of Eql Strains Results Glycerol Stocks of Stig117 Transformation of Stig117 with Eql Plasmids with Plating of Stig118 Stig123
23-06-08 Re Transformation of Donor Re Plating of Transformations from 23-06-07 Screening of Assembly Colonies Part 1
23-06-09 Screening of Assembly Colonies Part 2 Inoculation of Successful Assembly Strains
23-06-11 Inoculations of Strains in Eql Assay 2 Inoculations of Some Assembly Colonies
Week 3
23-06-12 Conjugation Prep of Donor and Recipient Strains for Eql Integrase Assay Miniprep of Gga Assemblies for Sequencing Glycerol Stocks PCR Diagnostic of Gga Assemblies Plasmidsaurus Sequencing
23-06-13 Conjugation Inoculation Plating of Strains for Eql Integrase Assay WPS Analysis Age of PCR Screening PCR Diagnostic of Assembly Plasmids Control Gga A111
23-06-15 Diagnostic PCR Age of Gga Controls Glycerol Stocks of Conjugated Recipients
23-06-16 PCR Amplification and Purification of A B C Backbone Gibson Assembly Module A Plasmid
23-06-17 Transformation of Gibson Assembly Entry Vectors
Week 4
23-06-19 PCR Colony Screening of Ga Plasmid Transformations
23-06-20 Repeat of PCR Colony Screening Amplification of Pz B and Pz C Gga of Modular Plasmids and Transformation of Gga S
23-06-21 Miniprep of Original Entry Vectors Plating of Modular Ggas Age on Gga PCR Assemblies Sanger Sequencing of Gibsons
23-06-22 Miniprep of Original Entry Vectors Gga Controls and PCR Screening Using Gga Assemblies
23-06-23 Miniprep of Original Entry Vectors Troubleshooting
23-06-24 Miniprep of Ga Entry Vectors for Sequencing
Week 5
23-06-26 Gga for BSAI Site Assay PCR PCR of All Modular Gga S Re-Send Gibsons Pcargo and Equol Strains for Sanger Sequencing
23-06-28 Enzyme Digestion Assay Gga Junction Assay Gibson Amplicon Creation EQL Calibration Curve
23-06-29 Enzyme Digestion Assay Retry Gga Amplicon Purification Retry Gga Amplicon PCR EQL Calibration Curve Analysis
23-06-30 Gibson Assembly and Transformation of WGA Modules EQL Calibration Analysis
23-07-01 PCR Colony Screening of GA Modules
Week 6
23-07-03 Mini-prep and WPS of Inoculated Colonies Diagnostic PCR on GA Products Feeding Experiment
23-07-04 Re-GA of Modular Plasmids, Extra DpNI Digest of PCARGO, Transform Initial GA Modular Plasmids, Trial Run of Equol Feeding Experiment
23-07-05 Plating of Re-GA Modular Plasmids and Re-electroporation of OG-GA-S, Colony PCR Junction Screening of OG-GA-S, Equol Feeding Experiment, and Extraction Positive Control
23-07-06 Colony PCR Screening of Re-GA Modules, Amplification of GGA Amplicon, Equol Feeding, and Extraction Experiments (Round 1)
23-07-07 PCR Purification Assay, Mini-prep of Potential Colonies, Test GGA Transformation, Equol Feeding Experiment
23-07-08 Colony Junction Screening, Equol Feeding Experiments
23-07-09 Colony Junction Screening, Test GA
Week 7
23-07-10 (A) GGA Amplification, GA Attempt 1, Colony Screening Retry
23-07-13 Mini-prep, Glycerol Stock, Successful GA and GGA3 for Sequencing, Transformation of Test GGA
23-07-14 WPS Analysis of Sequenced Modular Plasmids B1-M and Full GGA, Junction Screening, Conjugation for EQL Feeding
23-07-15 Mini-prep of Remaining Modular Plasmids for Sequencing, B1-M Junction Screening
23-07-16 More B1-M Colony PCR Screening
Week 8
23-07-17 Sending More Plasmids for WPS Backup, Colony PCR Screen, Daidzein Concentration Optimization
23-07-18 WPS Analysis, Electroporation of STIG117 with Correct Module Plasmids, IsoLig and Lig, DZN Standard Curves, Conjugation of P.utida Inducer Experiment, Equol
23-07-20 Conjugation of Modular Plasmid Strains into DH10LP, Mini-prep of GA Module Strains, Transformation of ASD with All B1-G3 Plasmids, Standard Curves for Intermediates, Extraction, LCMS for Validation of Initial Equol Experiment
23-07-21 Amplification of Test Run, Full Assembly from Modular Plasmids, Inoculations
23-07-22 Plating of Final Module Strains, Full Assembly Amplification Optimization, GA of Full Assembly
23-07-23 Test Feeding Experiment, Full Assembly PCR Colony Screen
Week 9
23-07-24 Homolog Optimization, LCMS, Begin Final Daidzein Plasmid Construction, LC-MS of Equol Validation Experiment
23-07-25 Final Daidzein Plasmid Construction, Transformation, and Plating
23-07-26 Final Daidzein Plasmid Construction, Colony Screen and Inoculation, Inoculation of STIG117
23-07-27 Final Daidzein Plasmid Construction, Mini-prep and WPS, Electroporation of STIG117 with Final Daidzein Plasmid
Week 10
23-07-28 Inoculation of Transformed STIG117 for Glycerol Stock
23-07-31 Colony PCR of Full Assembly, Extraction of Modular Feeding, LC-MS on Daidzein Concentration Curve
23-08-01 LC-MS on Equol Strains and Modular Strains, Homolog Optimization
23-08-02 Induction and Feeding for Equol in Different Strains Experiment
23-08-04 LC-MS of Nissle vs. DH10B Equol Strains and Thorough Inventory