Team:NYMU-Taipei/Notebook
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AFFILIATIONS & ACKNOWLEDGMENT
Protocol
Nitrogen-starvation-related experiments
PCC 6803 gDNA extractionDownload the file
NrtA expressionDownload the file
endolysin constructDownload the file
holin constructDownload the file
endolysin-holin constructDownload the file
endolysin-holin-NrtA constructDownload the file
Timeline
2017-06-19 prepare stock1 and stock5
2017-06-20 (1) reprepare stock5
(2) prepare BG11 medium
(3) unfreeze Synechocystis sp. PCC6803 and start to cultivate in BG11
2017-06-29 (1) prepare Pseudomonas aeruginosa growing medium
(2) activate Pseudomonas aeruginosa and start to cultivate in Petri dish
(3) subculture Synechocystis sp. PCC6803
2017-07-02 extract Pseudomonas aeruginosa plasmid and freeze into refrigerator
2017-07-03 proliferate Pseudomonas aeruginosa and freeze into refrigerator
2017-07-04 (1) prepare BG11 medium
(2) proliferate Chlorella vulgaris in Petri dish and centrifuge tube
2017-07-05 to 07-10 cultivate Synechocystis sp. PCC6803 and Chlorella vulgaris in incubator
2017-07-11 (1) extract Synechocystis sp. PCC6803 plasmid
(2) prepare LB containing ampicillin and chloramphenicol and prepare Petri dish
2017-07-12 transform BBa_K112806, BBa_K737007, BBa_K112000, BBa_J04450, BBa_K1356003, BBa_K1356004, and BBa_K356005 into competent cell
2017-07-13 (1) proliferate 07-12 transformed E.coli
(2) proliferate Chlorella vulgaris
(3) proliferate Synechocystis sp. PCC6803
(4) activate Synechococcus elongatus PCC7942
2017-07-14 (1)extract 07-13 proliferated E.coli plasmid
(2) digestion: BBa_K112806, BBa_K737007, BBa_K112000, BBa_J04450, BBa_K1356003, BBa_K1356004, and BBa_K1356005
(check the correction of transformed E.coli)
2017-07-17 (1) digestion again: BBa_K1356005, BBa_K112806, and BBa_K112000
(2) retransform BBa_K112806 and BBa_K112000
2017-07-18 (1) proliferate BBa_K112806 transformed E.coli (BBa_K112000 failed)
(2) subculture Synechococcus elongatus PCC7942
(3) cultivate Synechocystis sp. PCC6803 in BG11
2017-07-19 (1) extract 07-18 BBa_K112806 transformed E.coli plasmid
(2) digestion check
(3) save BBa_K112806 transformed E.coli
(4) save 07-18 Synechocystis sp. PCC6803
2017-07-20 (1) activate Rhizobium etli in PY medium
(2) dilute primer 001~008
(3) run NrtA and terminator PCR (failed)
2017-07-21 to 07-23 cultivate Chlorella vulgaris again for measuring growth curve
2017-07-24 (1) prepare Rhizobium etli liquid culture
(2) activate Synechococcus elongatus PCC7942
2017-07-25 (1) extract Synechocystis sp. PCC6803 genomic DNA
(2) run NrtA PCR
2017-07-26 (1) extract Rhizobium etli genomic DNA
(2) run NrtA and terminator PCR
(3) run NrtA+ terminator fusion PCR (failed)
2017-07-27 (1) run NrtA+ terminator fusion PCR again
(2) purify the successful fusion product
(3) digestion check
2017-07-28 run the whole NrtA cloning procedure again, using KOD polymerase
(1) run NrtA and terminator PCR
(2) digestion check NrtA and terminator and purify the PCR product
(3) run NrtA and terminator fusion PCR
(4) digestion check and purify the fusion PCR product (failed)
2017-07-29 (1) run NrtA and terminator fusion PCR again
(2) digestion check and purify the fusion PCR product
(3) ligation: backbone(psb1C3) and promoter, RBS, NrtA, and terminator
(4) transformation in to E.coli
2017-07-30 cultivate the transformed E.coli in liquid culture
2017-07-31 (1) extract 07-30 transformed E.coli plasmid
(2) restriction enzyme check (failed)
2017-08-04 Digest check: BBa_K112806
2017-08-05 Chlorella Vulgaris growth curve measuring
2017-08-06 PCR(Dream Taq): Sucide mechanism-BBa_K112806, K737007, J04450, K112000
2017-08-07 (1) PCR(KOD plus): Sucide mechanism-BBa_K112806, K737007, J04450, K112000
(2) NrtA- annealing, PNK, ligation, transformation
2017-08-08 Suicide mechanism-Annealing, cloning, ligation, transformation
2017-08-09 Extraction of Synechoccocus PCC7942 genomic DNA
2017-08-10 (1) Chlorella Vulgaris growth curve measuring
(2) Fusion PCR: NrtA + terminator
(3) Digestion check: NrtA cut with BamH1
(4) Subculture S.elongatus PCC7942
2017-08-11 (1) Ligation: NrtA + terminator + promoter + RBS
(2) Ligation: BBa_K737007+K112806, J04450+K112000 (Endolysin and Holin)
(3) Transformation of Endolysin and Holin
(4) Measure nitrate and nitrite concentration of Chlorella Vulgaris
(5) OD value measurement of Chlorella Vulgaris and S.elongatus PCC7942
2017-08-12 (1) Measure nitrate and nitrite concentration of Chlorella Vulgaris
(2) OD value measurement of Chlorella Vulgaris and S.elongatus PCC7942
2017-08-13 (1) Transformation: BBa_K274002 and K1152008 into E. coli
(2) Measure nitrate and nitrite concentration of Chlorella Vulgaris
(3) OD value measurement of Chlorella Vulgaris and S.elongatus PCC7942
2017-08-14 (1) Colony PCR: K1152008
(2) Suicide mechanism- PCR, annealing, cloning, ligation, transformation
(3) NrtA-PCR, annealing, cloning, ligation, transformation
(4) Liquid culture-K1152008
2017-08-15 (1) Measure nitrate and nitrite concentration of Chlorella Vulgaris
(2) OD value measurement of Chlorella Vulgaris and S.elongatus PCC7942
(3) Suicide mechanism- final annealing, cloning, ligation, transformation
(4) Plasmid extraction and RE check: k1152008
2017-08-16 (1) Measure nitrate and nitrite concentration of Chlorella Vulgaris
(2) OD value measurement of Chlorella Vulgaris and S.elongatus PCC7942
(3) PCR(Dream Taq): PrbCL and indC
2017-08-18 (1) Suicide mechanism- PCR, annealing, cloning, ligation, transformation
(2) PCR(KOD plus): PrbCL and indC
2017-08-19 (1) Suicide mechanism- PCR, annealing, cloning, ligation, transformation
(2) Inoculate E. coli containing pBAD18
(3) Fusion PCR: PrbCL+indC
2017-08-22 (1) PCR: Lycopene and MelA
(2) Fusion PCR: PrbCL+indC
2017-08-24 (1) Liquid culture: BBa_K274002
(2) Extract PCC7942 genomic DNA
(3) Inoculate E. coli containing PBR322
(4) Transformation: BBa_B0015 into E. coli
2017-08-25 (1) Endolysin- PCR, annealing, cloning, ligation, transformation
(2) Liquid culture: PBR322
(3) Liquid culture: B0015
2017-08-26 (1) Plasmid extraction: endolysin
(2) Plasmid extraction: PBR322
(3) Plasmid extraction: B0015
2017-08-27 (1) PCR: Lycopene and MelA
(2) Digestion check: PBR322
(3) Digestion check: B0015
(4) pigment backbone primer design
(5) PCR (Dream Taq): CrtZ
(6) Plasmid extraction: endolysin
(7) Digestion check: endolysin
2017-08-28 (1) Colony PCR: NrtA
(2) Liquid culture: NrtA
(3) Sequencing: endolysin
(4) Fusion PCR: PrbcL+IndC
(5) PCR(Dream taq): Holin(kit)
(6) Check pBR322 concentration and transform pBR322
2017-08-29 (1) Nile red oil measuring of Chlorella Vulgaris (~0901)
(2) Plasmid extraction: NrtA
(3) Holin construction
(4) sequencing: NrtA
2017-08-30 (1) PCR (Dream Taq): Ampicillin resistance, 5NSII, 3NSII, B0015, and PBR322 ori
(2) Endolysin construct finish
(3) Plasmid extraction: pBR322
(4) Restriction enzyme check: pBR322
(5) Backbone construct-3NSII, 5NSII,AMP, B0015, and ORI (PCR: dream taq)
2017-08-31 (1) PCR (KOD plus): Ampicillin, 5NSII, 3NSII, B0015, and PBR322 ori
(2) PCR (Dream Taq): PBR322 ori and B0015
(3) pSB1C3-NrtA construct finish
(4) Backbone construct-3NSII, 5NSII, AMP, B0015, and ORI (PCR: KOD)
(5) Transform: BBa_K112000
(6) LBA: glucose + CM (0.3%)
2017-09-01 (1)Fusion PCR: Ampicillin resistance, B0015
(2) Three pieces PCR: 5NSII, 3NSII, and PBR322 ori
2017-09-02 (1) Ligation: Ampicillin resistance, B0015+ 5NSII, 3NSII, and PBR322 ori
(2) Transformation: pPIGBACK (pigment plasmid backbone)
(3) PCR (Dream Taq): CrtZ and PrbCL
2017-09-03 (1) Colony PCR: pPIGBACK
(2) Liquid culture: pPIGBACK
(3) PCR (KOD): CrtZ and PrbCL
2017-09-04 (1) Plasmid extraction: pPIGBACK
(2) Digestion check: pPIGBACK
(3) Fusion PCR: CrtZ+ PrbCL
2017-09-05 (1) pPIGBACK construct finish
(2) Fusion PCR: PrbCL+IndC
(3) Ligation: CrtZ+ PrbCL with pPIGBACK
(4) Transformation: CrtZ construct
2017-09-06 (1) Colony pcr: CrtZ construct
(2) Liquid culture: CrtZ
(3) Lycopene primers design
(4) IndC primers redesign
2017-09-07 (1) plasmid extraction: CrtZ-Ppigback
(2) restriction enzyme check: CrtZ-pPIGBACK
2017-09-08 (1) transform CrtZ-Ppigback into PCC7942
(2) nitrate and nitrite adsorption assay of Chlorella vulgaris (co-coculture with NrtA)
(3) transform suicide mechanism construct (LBA+glucose 0.3%)
2017-09-09 (1) nitrate and nitrite adsorption assay of Chlorella vulgaris (co-coculture with NrtA)
(2) fusion PCR: PrbcL+indC
2017-09-10 indC construct (~transformation)
2017-09-19 (1) Endolysin-Holin construct
(2) Site-directed primers design
2017-09-20 (1) Liquid culture: BBa_J04450
(2) Liquid culture: CrtZ-PCC7942 clones
2017-09-21 Plasmid extraction: J04450, CrtZ-PCC7942
2017-09-22 Site-directed PCR: 3NSII
2017-09-23 Site-directed-3NSII construct finish
2017-09-24 (1) plasmid extraction: Site-directed-3NSII
(2) Site-directed PCR: 5NSII-1
(3) Psb1c3-PrbcL construct finish
2017-09-25 (1) Psb1c3-NrtA construct finish
(2) colony PCR: Psb1c3-PrbcL
(3) Liquid culture: Psb1c3-PrbcL
(4) IndC construct
2017-09-26 (1) Plasmid extraction and restriction enzyme check: Site-directed-5NSII-1
(2) Liquid culture: Psb1c3-NrtA
(3) Colony PCR: IndC
(4) Psb1c3-3NSII construct finish
(5) Site-directed PCR: 5NSII-2
2017-09-27 (1) Psb1c3-NrtA construct finish
(2) Plasmid extraction and restriction enzyme check: IndC
(3) Plasmid extraction and restriction enzyme check: IndC
(4) Psb1c3-3NSII construct finish Plasmid extraction and RE check: Site-directed 5NSII-2
(5) Site-directed PCR: AmpR
2017-09-28 (1) Plasmid extraction and RE check: Site-directed-AmpR
(2) Plasmid extraction and RE check: Psb1c3-5NSII
(3) Psb1c3-3NSII(SD) finish
(4) Liquid culture: Psb1c3-PrbcL
2017-09-29 (1) Psb1c3-5NSII construct finish
(2) Psb1c3-5NSII-ORI-3NSII construct finish
(3) Colony PCR: Psb1c3-PrbcL- IndC
(4) Colony PCR: Psb1c3-PrbcL- IndC
(5) Psb1c3-PrbcL(SD) construct finish
(6) Psb1c3-PrbcL-IndC construct finish
(7) Site-directed: amp finish
(8) transform CrtZ-pPIGBACK into PCC7942
2017-09-30 (1) Psb1c3-PrbcL(SD) finish
(2) Liquid culture: CrtZ-pPIGBACK
