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Revision as of 03:33, 17 September 2017
Protocol
NrtA expression
Suicide gene expression
OD measurement
Timeline
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)
