Team:iBowu-China/NoteBook
iBowu-China has compiled an account of everything that has happened in the lab over the summer. We are excited to show you how busy we have been in iGEM 2019! We believe that sharing our notes in labs will convey the passion and dedication of our teammembers, as we continuously push our limits to achieve high quality results.
Date:20190323 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Harry Fang, Annie Xian Goals: Construction of PSB3K3-LuxR-sfGFP plasmid Experiment steps and results: -PCR amplification of PSB3K3-LuxR-sfGFP plasmid -Gel Electrophoresis and extraction of PCR products -Failed PCR amplification (Wrong length for LuxR sequence)
Date:20190324 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Harry Fang, Annie Xian Goals: Construction of PSB3K3-LuxR-sfGFP plasmid Experiment steps and results: -PCR amplification of PSB3K3-LuxR-sfGFP plasmid -Gel Electrophoresis and extraction of PCR products -Successful PCR amplification -Gibson assembly of PSB3K3-LuxR-sfGFP plasmid (8 μl system, 30min at 50 degrees)
Date:20190330 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Harry Fang, Annie Xian Goals: Construction of PSB3K3-LuxR-AMP plasmid Experiment steps and results: -PCR amplification of PSB3K3-LuxR-AMP plasmid -Gel Electrophoresis and extraction of PCR Products -Failed PCR amplification
Date:20190331 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Harry Fang, Annie Xian Goals: Construction of PSB3K3-LuxR-AMP plasmid Experiment steps and results: -PCR amplification of PSB3K3-LuxR-AMP plasmid -Gel Electrophoresis and extraction for PSB3K3-LuxR -AMP -Successful PCR amplification -Gibson assembly of the PSB1K3-LuxR-AMP plasmid (8 μl system, 30min at 50 degrees) -Transfection of the plasmid into E. Coli competent cell culture
Date:20190420 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Harry Fang, Annie Xian, Peter Qi Goals: Construction of PSB3K3-LuxR-AMP plasmid Experiment steps and results: -PCR Products prepared -Gibson assembly of PSB3K3-LuxR-AMP plasmid (8 μl system, 30min at 50 degrees) -Transfection of PSB3K3-LuxR-AMP plasmid into E. Coli competent cell culture
Date:20190421 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Harry Fang, Annie Xian, Peter Qi Goals: -Sequencing of successfully grown bacterial colonies -Construction of PSB3K3-promoter plasmid Experiment steps and results: -PCR products prepared -Gibson Assembly of PSB3K3-promoter plasmid (8 μl system, 30min at 50 degrees) -Transfection of the constructed plasmid into E. Coli competent cell culture -Picked up four positive bacterial colonies for sequencing at BGI
Date:20190512 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Rouxuan Yu, Annie Xian, Peter Qi Goals: Construction of PSB3K3-LuxR-AiiA, PSB3K3-LuxR-LacZ, PSB3K3-LuxR-sfGFP plasmids Experiment steps and results: -PCR amplification of PSB3K3-LuxR-AiiA, PSB3K3-LuxR-Lacz, and PSB3K3-LuxR- sfGFP plasmids -Gel electrophoresis and extraction of plasmids -Successful amplification of all plasmids
Date:20190713 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Ian Liu, Annie Xian Goals: Construction of PSB3K3-CarR-AMP, PSB3K3-CarR-LacZ, PSB3K3-CarR-sfGFP, PSB3K3-CarR-AiiA plasmids Experiment steps and results: -PCR amplification of all sequences -Gel Electrophoresis and extraction of all plasmids -Successful amplification of PSB3K3-CarR-sfGFP plasmid -Failed amplification of PSB3K3-CarR-AMP, PSB3K3-CarR-LacZ, PSB3K3- CarR-AiiA plasmids -Gibson assembly of PSB3K3-CarR-sfGFP plasmid(8 μl system, 30min at 50 degrees) -Transfection of PSB3K3-CarR-sfGFP plasmid into E. Coli. -No growth of E. Coli colonies
Date:20190714 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Ian Liu, Annie Xian Goals: Construction of plasmid PSB3K3-CarR-AMP plasmid Experiment steps and results: -Restarting experimental progress from July 15th after discussion regarding our previous failures
Date:20190715 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Ian Liu, Annie Xian Goals: Construction of PSB3K3-CarR-AMP, PSB3K3-CarR-LacZ, PSB3K3-CarR-sfGFP, PSB3K3-CarR-AiiA plasmids Experiment steps and results: -PCR amplification of all plasmids -Gel Electrophoresis and extraction of all plasmids -Failed PCR amplification of all plasmids -Plasmid backbone too long, redesign of PCR primers
Date:20190716 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Ian Liu, Harry Fang Goals: Construction of PSB3K3-CarR-AMP, PSB3K3-CarR-LacZ, PSB3K3-CarR-sfGFP, PSB3K3-CarR-AiiA Experiment steps and results: -PCR amplification of all plasmids -Gel Electrophoresis and extraction of all plasmids -Failed PCR amplification of all plasmids -Redesign of primers
Date:20190718 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Harry Fang, Ian Liu, Annie Xian Goals: Construction of PSB3K3-CarR-AMP, PSB3K3-CarR-LacZ plasmids Experiment steps and results: -PCR amplification of both plasmids *(Each plasmid amplified from two different ends marked as N1 and N2, N1 being the kana antibiotic resistance gene) -Gel Electrophoresis and extraction of both plasmids -Both plasmids amplified successfully -Gibson assembly of both plasmids (8 μl system, 30min at 50 degrees) -Transfection of both plasmids into E. Coli competent cell culture
Date:20190719 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Ian Liu, Annie Xian Goals: Construction of PSB3K3-CarR-AMP, PSB3K3-CarR-LacZ plasmid Experiment steps and results: -Successful E. Coli growth for both plasmids -PCR amplification of E. Coli colonies -Amplified colonies sent away for sequencing at BGI -Correct sequence for both plasmids
Date:20190722 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Harry Fang, Annie Xian Goals: Construction of PSB3K3-CarR-sfGFP, PSB3K3-CarR-AiiA plasmids Experiment steps and results: -PCR amplification of both plasmids *(Each plasmid amplified from two different ends marked as N1 and N2, N1 being the kana antibiotic resistance gene) -Gel Electrophoresis and extraction of both plasmids -Successful amplification of both plasmids -Gibson assembly of both plasmids (8 μl system, 30min at 50 degrees) -Transfection of both plasmids into E. Coli competent cell culture -No growth of E. Coli colonies
Date:20190724 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Annie Xian, Harry Fang Goals: Construction of PSB3K3-CarR-sfGFP, PSB3K3-CarR-AiiA plasmids Experiment steps and results: -PCR amplification of both plasmids *(Each plasmid amplified from two different ends marked as N1 and N2, N1 being the kana antibiotic resistance gene) -Gel Electrophoresis and extraction of both plasmids -Successful amplification of both plasmids -Gibson assembly of both plasmids (8 μl system, 30min at 50 degrees) -Transfection of both plasmids into E. Coli competent cell culture -No growth of E. Coli colonies
Date:20190725 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Annie Xian, Harry Fang Goals: Construction of PSB3K3-CarR-sfGFP, PSB3K3-CarR-AiiA plasmids Experiment steps and results: -Reconfiguration of petri dishes used for competent cell culture growth -PCR amplification of both plasmids *(Each plasmid amplified from two different ends marked as N1 and N2, N1 being the kana antibiotic resistance gene) -Gel Electrophoresis and extraction of both plasmids -Successful amplification of both plasmids -Gibson assembly of both plasmids (8 μl system, 30min at 50 degrees) -Transfection of both plasmids into E. Coli competent cell culture -No growth of E. Coli colonies -Used a competent E. Coli cell culture to test for cell death -Failed experiment likely due to death of competent cells used for transfection
Date:20190726 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Peter Qi, Harry Fang Goals: Construction of PSB3K3-CarR-sfGFP, PSB3K3-CarR-AiiA plasmids, Cell-Free synthesis of previously successfully constructed plasmids Experiment steps and results: -No E. Coli colony growth even when competent E. Coli cells are active -Extraction of previously constructed and sequenced plasmids (PSB3K3-LuxR-AiiA#1, PSB3K3-LuxR-sfGFP#1, PSB3K3-LuxR-sfGFP#4, PSB3K3-LuxR-AMP#1, PSB3K3- LuxR-AMP#4) for Cell-Free synthesis -Failed construction for both plasmids led us to buy new competent transfection cells from another source for future experiments
Date:20190727 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Peter Qi Goals: Construction of PSB3K3-CarR-sfGFP, PSB3K3-CarR-AiiA plasmids Experiment steps and results: -PCR amplification of both plasmids *(Each plasmid amplified from two different ends marked as N1 and N2, N1 being the kana antibiotic resistance gene) -Gel Electrophoresis and extraction of both plasmids -Successful amplification of both plasmids -Gibson assembly of both plasmids (8 μl system, 30min at 50 degrees) -Transfection of both plasmids into E. Coli competent cell culture -Successful growth of E. Coli colonies
Date:20190728 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Peter Qi Goals: Construction of PSB3K3-CarR-sfGFP, PSB3K3-CarR-AiiA plasmids, preparation of PSB3K3-LuxR-AiiA, PSB3K3-LuR-sfGFP plasmids for Cell-Free synthesis Experiment steps and results: -PCR amplification of E. Coli colonies for both plasmids -Amplified plasmids sent to BGI for sequencing -Transfer of previously constructed PSB3K3-LuxR-AiiA, PSB3K3-LuxR-sfGFP plasmids into LB solution for overnight incubation
Date:20190729 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Peter Qi Goals: Extraction of incubated PSB3K3-LuxR-AiiA, PSB3K3-LuxR-sfGFP plasmids Experiment steps and results: -Both plasmids successfully extracted from E. Coli colonies
Date:20190730 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Peter Qi Goals: Demonstration of Cell-Free synthesis and incubation of previously constructed plasmids Experiment steps and results: -PSB3K3-LuxR-AiiA#1, PSB3K3-LuxR-sfGFP#1, PSB3K3-LuxR-sfGFP#4, PSB3K3- LuxR-AMP#1, PSB3K3-LuxR-AMP#4 Plasmids transferred into LB solution for overnight incubation
Date:20190731 Location: Haidian Experimental Middle School iGEM Laboratory Persons involved: Peter Qi, Harry Fang, Linda Liu, Hedy Zhu Goals: Extraction and cell-free synthesis of PSB3K3-LuxR-AiiA#1, PSB3K3-LuxR-sfGFP#1, PSB3K3-LuxR-sfGFP#4, PSB3K3-LuxR-AMP#1, PSB3K3-LuxR-AMP#4 plasmids Experiment steps and results: -Successful extraction of all incubated plasmids -Other plasmids stored in – 20-degree Celsius fridge as spare parts -Cell-Free synthesis of extracted plasmids -Failed synthesis -Re-extraction of incubated plasmids successful -Second attempt of Cell-Free synthesis failed