Team:Lanzhou/Notebook

Lanzhou

Lanzhou2017

April

  • Week 1 Apr 1 keyboard_arrow_down
    • • Establish research project in group meeting.
  • Week 2 Apr 2 ~ Apr 8 keyboard_arrow_down
    • • Prepare for experiment and confirm members of the division of labor.
  • Antibiotic(Amp,Kana,Chl,Tet).
  • prepare 5M IPTG.
  • prepare double distilled water, centrifuge tube, PCR tube sterilization, of alcohol cotton.
  • Preparation of competent state.
  • Week 3 Apr 9 ~ Apr 15 keyboard_arrow_down
    • • New members acquaint the laboratory,and do some basic experimental to prepare for the IGEM.
  • Week 4 Apr 16 ~ Apr 22 keyboard_arrow_down
    • • Verify the plasmids and primer in group meeting.
  • Week 5 Apr 23 ~ Apr 29 keyboard_arrow_down
    • • Gene synthesis

May

  • Week 1 Apr 30 ~ May 6 keyboard_arrow_down
    • • Direct mutagenesis
  • Site-directed mutagenesis Lon1, Lon2.and run the gel to proving the verification of the mutation.
    • • Extract plasmids
    • Digestion
  • Digested pUC19 (EcoRI, PstI), LacI and gel extraction.
  • Week 2 May 7 ~ May 13 keyboard_arrow_down
    • • Site-directed mutagenesis Lon1, Lon2.
  • make bacterium Lon1, Lon2, pET28a (+), pUc19, pETDuet amplified.
    • • Transformation
  • Transform the plasmids into E.Coli DH5a.
    • • Culture
  • pick a single clone on the transformed plate and culture in fluid culture medium.
  • Week 3 May 14 ~ May 20 keyboard_arrow_down
    • • Ligation
  • Use protocol of the Quick Connect Enzyme Kit to connect pUC19+GFP，pUC19+LacI.
    • • Transformation
  • Transform the mutation,connect product and L4440 plasmids.
  • Week 4 May 21 ~ May 27 keyboard_arrow_down
    • • Plasmids extraction
  • Extract the plasmids of Gmix and P with plasmids Mini Kit,then run the gel electrophoresis (Figure 1).
    • Gel electrophoresis
  • run the gel electrophoresis of Ga(1~5) (Figure2).
  • run the gel electrophoresis of Gb (1~5) (Figure3).
    • • Gel purification of Ga（Figure4）and Gb (Figure4).
    Figure 1.
    Figure 2.
    Figure 3.
    Figure 4.
  • Week 5 May 28 ~ Jun 3 keyboard_arrow_down
    • • Digestion
  • digest Gain E/S,Gbin E/S.
    • • Ligation
  • Ligate Ga with pUC19,Gb with pUC19,Ga+Gb with pUC19.
    • • Transformation
  • Transform Ga+pUC19 into DH5a,Gb+pUC19 into DH5a,Ga+Gb+pUC19 into DH5a.
    • • Run gel
  • Extract pUC19 into 6 tubes.
  • Gel purification of puc19 (Figure 6&7), analyse the failure of digestion.
    • `
    Figure 6.
    Figure 7.
    • Ligation
  • Ligated GaE/P with Puc19 E/P, GbE/P with pUC19 E/P, GaE/S+GbX/P with pUC19 E/P.then Transfer ligated product into the plate,and Cultivate them overnight.

June

  • Week 1 Jun 4 ~ Jun 10 keyboard_arrow_down
    • • PCR
  • PCR Ga, Gb, P, then gel purification (Figure 8&9).
  • PCR protocol
    • Figure 8.
    Figure 9.
    • Plasmids extraction
  • Plasmids extraction of pUC19+Ga，pUC19+Gb.
  • Week 2 Jun 11 ~ Jun 17 keyboard_arrow_down
    • • Repeat-ligation
  • Ligate GaE/P with Puc19 E/P,GbE/P with pUC19 E/P,GaE/S+GbX/P with pUC19 E/P. • Gel purification
    • Repeat digestion
  • Repeat pUC19+Ga.E/S,pUC19+Gb.X/P. (Figure10)
    • • Digestion
  • Restriction digest P+EP6,ECR+EP10,ECR+EP6,SHT+EP6,SHT+EP8 (Figure11&12&13).
    • Figure 10.
    Figure 11.
    Figure 12.
    Figure 13.
    • Repeat PCR
  • Repeat Gb PCR then gel purification
  • Week 3 Jun 18 ~ Jun 24 keyboard_arrow_down
    • • Transformation
  • Transform ECR+L4440 and SHT+L4440into DH5a.
    • • Repeat PCR
  • repeat Gb PCR and P digest (Figure14&15).
    • Figure 14.
    Figure 15.
    • Ligation
  • ligate ECR+pUC19 (Figure16)
    • Figure 16.
  • Week 4 Jun 25 ~ Jul 1 keyboard_arrow_down
    • • Culture
  • Culture bacterium in glycerol:MIX,GMIX, B1.
    • • plasmids extraction
  • MIX,GMIX, B1.
    • • Digestion
  • Digest MIX in P/E product and gel purification.
    • • Transformation
  • Transform the produce into puC19,then pick the positive.

July

  • Week 1 Jul 2 ~ Jul 8 keyboard_arrow_down
    • • PCR
  • Do GMIX PCR
    • • Transformation
  • Transform PCR GMIX and into puC19.
  • Tansform RcsA，RBS，pUC19 into BL21.
    • • Digestion
  • L4440，GbEXSP，GaEXSP and B1EXSP pick the positive and digest.
  • GbEXSP，GaEXSP and B1EXSP.
    • • Plasmids extraction
  • GbEXSP，GaEXSP and B1EXSP extract plasmids.
  • Kil，GFP，RBS，OmpT.
  • Week 2 Jul 9 ~ Jul 15 keyboard_arrow_down
    • • Transformation:OmpT，pUC19.
  • Culture 17P: 06 (0.47), Ga, Ga2, Gb, Gb2, B1, B2, SHT, Ecr, Terminator, pUC19, RcsA, pSB3K3, pSB4K5.
  • PCR loop120
    • • plasmids extraction
  • 17P: 06 (0.47), Ga, Ga2, Gb, Gb2, B1, B2, SHT, Ecr, Terminator, pUC19, RcsA, pSB3K3, pSB4K5.
    • • Digestion
  • Ga, Ga2, Gb, Gb2, B1, B2, SHT, SHT2, Ecr, Ecr2, RcsA, pUC19，pSB3K3, pSB4K5, Loop120, Lysis1, lysis2, Terminator.
  • Week 3 Jul 16 ~ Jul 22 keyboard_arrow_down
    • • Ligation
  • Ga2+T——Ga2T
  • Gb2+T——Gb2T
  • B2+T——B2T
  • SHT2+T——S2T
  • Ecr2+T——E2T
  • Loop120+pUC19——Loop120
  • BBa_B0030+17P: 06 (0.47)——0.47x0.6
  • BBa_B0064+17P: 06 (0.47)——0.47x0.35
  • RcsA+T——RcsAT
  • Lysis1+pUC19——lysis1
  • Lysis2+pUC19——lysis2
    • • Plasmids extraction
  • Extract plasmids of pSB1T3，pSB1K3，pSB1C3，pSB1A3，SHT.
  • Week 4 Jul 23 ~ Jul 29 keyboard_arrow_down
    • • PCR
  • Ga2T,Gb2T,B2T,S2T,E2T,Loop120,RcsAT,lysis1,lysis2.
  • Prepare the transform cell of M bacterium,and all the plasmids need to transform would be gone and well.

August

  • Week 1 Jul 30 ~ Aug 5 keyboard_arrow_down
    • • Human practice
  • Confirm the form for human practice in group meeting.
  • Week 2 Aug 6 ~ Aug 12 keyboard_arrow_down
    • • Human practice
  • Design a questionnaire of transgenosis in public.
  • Week 3 Aug 13 ~ Aug 19 keyboard_arrow_down
    • • Human practice
  • Go to some schools advertise our work.
  • Week 4 Aug 20 ~ Aug 26 keyboard_arrow_down
    • • We went to FAFU for CCIC.
  • Week 5 Aug 27 ~ Sep 2 keyboard_arrow_down
    • • Gene synthesis
  • Synthesize the gene sequence act on arabidopsis thaliana.
    • • Vector construction
  • Construct 3hree vectors producing dsRNA (Ecr-L4440, ECRL, ECRN)
    • • Experiment on aphids.

September

  • Week 1 Sep 3 ~ Sep 9 keyboard_arrow_down
    • • Transformation
  • Transform carrier(Ecr-L4440, ECRL, ECRN) into E.coli M-JLAC19 (Dicer deficit type).
    • • Induce expression
    • Try to express dsRNA form trxzDP.
  • Week 2 Sep 10 ~ Sep 16 keyboard_arrow_down
    • • Induce expression
  • trxzDP:product dsRNA by T7 bi-directional promoter.
  • trxzL: product hpRNA by normal loop construction.
  • trxzN: product hpRNA by intron loop construction.
  • Week 3 Sep 17 ~ Sep 23 keyboard_arrow_down
    • • Induce expression
  • Ecr-L4440:product dsRNA by T7 bi-directional promoter.
  • ECRL:product hpRNA by normal loop construction.
  • Week 4 Sep 24 ~ Sep 30 keyboard_arrow_down
    • • Express of dsRNA and hpRNA.

October

  • Week 1 Oct 1 ~ Oct 7 keyboard_arrow_down
    • • Culture arabidopsis thaliana (Col 0)
    • Vector construction
  • Construct vectors of dsRNA which act on arabidopsis thaliana.
    • • Transformation
  • Transform carrier(trxzDP, trxzL, trxzN) into E.coli M-JLAC19 (Dicer deficit type).
    • • Induce expression
  • induce the M-JLAC19 express dsRNA.
  • Week 2 Oct 8 ~ Oct 14 keyboard_arrow_down
    • • Express dsRNA and hpRNA.
    • Prepare for the macro experiments validation.
  • Week 3 Oct 15 ~ Oct 21 keyboard_arrow_down
    • • Improve parts
    • Modelling work
  • Week 4 Oct 22 ~ Oct 28 keyboard_arrow_down
    • • Modelling work
    • Presentation work
  • Week 5 Oct 29 ~ Oct 31 keyboard_arrow_down
    • • Modelling work
    • Wiki & design work
    • Presentation work