Team ice-breaking.
Team discussion（Team name, uniform, logo...）.
Laboratory safety training.
Configuring the culture medium.
Inoculation of the Streptomyces rapamycinicus, 30℃.
Inoculation of the strain containing plasmid pKC1139.
Design the questionnaires.
Extract plasmid pKC1139 using a plasmid extract kit.
Team collaboration.
Amplify target DNA fragments by PCR.
PCR result:
Line 1, 3, and 5 are upstream homologous gene fragments, and line 2, 4, and 6 are downstream homologous gene fragments.
Line 1, 4 and 7 are plasmid pKC1139.
Line2,3,5,6,8,9,10 are double enzyme digested plasmid pKC1139 (EcoRI/HindIII).
Construct recombinant plasmids by Homologous reorganization.
Verification of the recombinant plasmids pKC-M271_ 14685/ M271_ 14690through colony PCR.
Inoculation of the strain containing correct plasmids.
Extract the plasmids pKC-M271_ 14685/ M271_ 14690 with a plasmids extraction kit.
Double enzyme digestion verification.
Line 1,3,5 are recombinant plasmid pKC-M271_ 14685/ M271_ 14690.
Line 2,4,6 are recombinant plasmid pKC-M271_14685/ M271_14690 digested with EcoR I/Hind III
Expert interview.
Transfer plasmids pKC-M271_ 14685/ M271_ 14690 into ET12567/pUZ8002 competent cells, and coat to LB plate containing antibodies, 37℃ incubate overnight.
Take fixed makeup photos.
Pick up single colonies and inoculate them in 4ml fresh culture medium,37℃ 220rpm overnight.
1. Transfer 1ml strain suspension into 50ml fresh culture medium, when OD600 is around 0.4-0.6, centrifuge and discard the medium, wash it twice with LB (no antibody contained) and resuspended it with 500ul culture medium.
2. Collect the Streptomyces rapamycinicus spore through 2×YT culture medium which was incubated on the plate.
3. Mix the E. coli suspension from step (1) and the spore suspension from step (2), coat onto M-ISP4 solid medium, and incubate at 30°C.
After incubated on the M-ISP4 solid medium for 16h, add antibodies, and incubate at 30℃ for 7 days.
PCR identification of colony.
Screening for single cross-over strains.
Filtrate single cross-over strains.
Carry out educational activities.
Expert interview.
Screening double cross-over strains.
The correct strain was streaked on the oat solid medium and cultured at 30°C for 7 days
Expert interview.
Writing implementation.
Fermentation culture of Streptomyces rapamycinicus.
Pick up colonies and inoculate them in 3ml fresh fermentation culture medium,28℃ 220rpm overnight.
Transfer 3ml strain suspension into 30ml fresh fermentation culture medium, culture at 28℃ 200rpm, and collect samples at 5d, 7d, 9d, and 11d.
Rapamycin production estimation by HPLC.
Write wiki.
Make a presentation video.
Prepare for presentation.
