Team:iBowu-China/NoteBook

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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.

Day1

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)

Day2

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)

Day3

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

Day4

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

Day5

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

Day6

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

Day7

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

Day8

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

Day9

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

Day10

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

Day11

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

Day12

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

Day13

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

Day14

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

Day15

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

Day16

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

Day17

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

Day18

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

Day19

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

Day20

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

Day21

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

Day22

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