Team:NCTU Formosa/calendar

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         <li><a href="https://2011.igem.org/Team:NCTU_Formosa/members">Members</a></li>
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         <li><a href="http://www.nctu.edu.tw/english/index.php">School</a></li>
         <li><a href="https://2011.igem.org/Team:NCTU_Formosa/gallery">Gallery</a></li>
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Revision as of 09:21, 3 October 2011



DateG1G2G2-2G2-1
4/26/2011colon PCR vioABCE+terminatordigest Ptet ES site, Ligate CrtEBI+Ptet +1K3 overnight
4/27/2011extract Plac plasmid, ligation Plac+37RBSPtet+crt EBI transform
4/28/2011Electrophoresis of vioABCE+terminator & incubate LB ; transform Plac+37RBSligate Ptet+crtEBI+1c3(zip) and transform
4/29/2011prepare plasmid of vioABCE+terminatorYesterday transform failed.ligate ptet+crtEBI+1c3 again.(non-purify 1C3)
4/30/2011ligate ptet+crtEBI+1c3(purify) overnight.
5/1/2011transform Plac+37RBSextract Ptet plasmid. Transform
Ptet+crtEBI+1c3
5/2/2011transform Ptet+vioABCE+terminator
5/3/2011Digest crtEBI XP and Ptet SP
5/4/2011 Ptet digest ES ; vioABCE+terminator digest XP ;ligation Ptet+vioABCE+terminatorLigate ptet(purify) Amp (digest SP)+crtEBI(purify)(digestXP). Ligate ptet(ES)+crtEBI(XP)+1c3(EP)
5/5/2011transform Ptet+vioABCE+terminatorTransform 5/4 two ligations.Ligate ptet(purify)Amp (SP)+crtEBI(purify)(XP).
5/6/2011Transform ptet(purify)Amp(SP)+crtEBI(purify)(XP).
5/7/2011incubate Ptet,CrtEBI
5/8/2011extract Ptet CrtEBI plasmid
5/10/2011Plac+37RBS colony PCR ; prepare plasmid of Plac+37RBS Ligate Ptet+CrtEBI+1c3(original method) and Ligate Ptet+CrtEBI+1c3(purify)
5/11/2011Ptet+vioABCE+terminator colony PCR ;Plac+37RBS extract plasmidTransform Ptet+CrtEBI+1c3(original method) ,Plac+CctEBIY,Plac+CrtEBI
5/12/2011ligation LuxR+terminatorPCR Ptet+CrtEBI+1c3CrtY point mutation PCR
5/13/2011transform LuxR+terminatorElectrophoresis.PCR Ptet+CrtEBI+1c3 againCrtY self-ligation
5/14/2011ligation LuxR+terminatorElectrophoresis.CrtY transform
5/15/2011transform LuxR+terminator
5/16/2011 LuxR+terminator colony PCR
5/17/2011 LuxR+terminator incubate LBPtet digest sp ,crtEBI digestXP (purify both),Ligation.
CrtY colonyPCRDigest C0012 ES & XP;
B0034 ES


5/18/2011prepare plasmid of LuxR+terminator ; ligation J24679+LuxR+terminator; prepare plasmid of Ptet & digest EP SP; incubateLB of k274003 Transform Ptet SP+CrtEBI XPLigase C0012+37RBS
+tetR+terminator & B0034+C0012
Digest B0034+GFP+terminator
5/19/2011ligation Ptet+vioABCE+terminator ; prepare plasmid of k274003 ; transform J24679+LuxR+terminatortransform C0012+37RBS
+tetR+terminator & B0034+C0012
5/20/2011 J24679+LuxR+terminator colony PCR ; incubate LB of Ptet CrtY colonyPCRPCR
C0012+37RBS
+tetR+terminator
5/21/2011prepare plasmid of Ptet ; ligation and transform J24679+LuxR+terminator ; ligation and transform Ptet+vioABCE+terminatordigest CrtY(S) →success
5/22/2011Ptet+vioABCE+terminator colony PCR dilute plasmid RBS+crtZ from 2011plate19B-I742158
5/24/2011digest CrtY(E,S) Terminator(E,X)
5/25/2011ligation and transform J24679+LuxR+terminator ligase CrtY+Terminator
5/26/2011think of new ligation and transform protocol of J24679+LuxR+terminator transform CrtY+Terminator
5/27/2011follow new protocol to ligation and transform protocol of J24679+LuxR+terminator (add 1mM ATP when ligation)
5/28/2011J24679+LuxR+terminator colony PCR & incubate LB
5/29/2011prepare plasmid of J24679+LuxR+terminator X2
5/30/2011prepare plasmid of J24679+LuxR+terminator X5
5/31/2011check J24679+LuxR+terminator plasmid
6/1/2011transform RBS+crtZcolony PCR CrtY+Terminator
6/2/2011colony PCR RBS+crtZ
electrophoresis check
sequencing RBS+crtZ
6/3/2011ligase CrtY+Terminator
6/4/2011transform CrtY+Terminator
6/7/2011sequencing result RBS+crtZ → successs
incubate RBS+crtZ in LB-medium		colony PCR CrtY+Terminator →success
6/8/2011extract plasmid RBS+crtZ
6/10/2011digest RBS+crtZ(ES)(anti-A)
digest terminator-J61048(XP&EX)(anti-A)
digest PSB1K3(EP)(anti-K)
ligate 1.RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K)
2.RBS+crtZ(ES)+terminator(EX)(anti-A)
6/11/2011transform 1.RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K)
2.RBS+crtZ(ES)+terminator(EX)(anti-A)		incubate 1.37RBS 2.CrtY_Terminator 3.psb1k3 4.psb1c3
6/12/2011only RBS+crtZ(ES)+terminator(EX)(anti-A) grown
colony PCR above-mentioned
electrophoresis check
sequencing RBS+crtZ(ES)+terminator(EX)(anti-A)
digest RBS+crtZ(ES)(anti-A)again
digest terminator-J61048(XP)(anti-A)again
digest PSB1K3(EP)(anti-K)again
ligate RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K)again		extract plasmid digest 1.37RBS(E,S).(S,P) 2.CrtY_Terminator(X,P) 3.psb1c3(E,P) ligase 37RBS+CrtY_Terminator
6/13/2011transform RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K)againtransform 37RBS+CrtY_Terminator
6/17/2011sequencing result RBS+crtZ(ES)+terminator(EX)(anti-A) → successs
6/23/2011digest 37RBS and kivd ; incubate LB of B0030,B0032,B0034
6/24/2011prepare plasmid of B0030,B0032,B0034 & check ; ligation 37RBS+kivdincubate RBS+crtZ+terminator(anti-A) in LB-medium
6/25/2011digest ilvC,ilvD,malss,B0030,B0032,B0034extract plasmid RBS+crtZ+terminator(anti-A)
digest RBS+crtZ+terminator(XP)(anti-A)
digest pcI(ES&SP)(anti-A)
digest PSB1K3(EP)(anti-K)
ligate 1.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)
2.pcI(SP)+RBS+crtZ+terminator(XP)(anti-A)		colony PCR 37RBS+CrtY_Terminator→success
6/26/2011check digest producttransform 1.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) 2.pcI(SP)+RBS+crtZ+terminator(XP)(anti-A)
6/27/2011only pcI(SP)+RBS+crtZ+terminator(XP)(anti-A) grown
colony PCR can't examine pcI(SP)+RBS+crtZ+terminator(XP)(anti-A)
because pcI is too small(49bp)
incubate pcI(SP)+RBS+crtZ+terminator(XP)(anti-A) in LB-medium
6/28/2011extract plasmid pcI(SP)+RBS+crtZ+terminator(XP)(anti-A)
digest RBS+crtZ+terminator(XP)(zip)(anti-A)again
digest pcI(ES&SP)(anti-A)again
digest PSB1K3(EP)(anti-K)again
digest PSB1C3(EP)(anti-C)again
ligate 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)again
2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)again
3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C)again
4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C)again
5.pcI(SP)+RBS+crtZ+terminator(XP)(zip)(anti-A)again
6/29/2011transform 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)again
2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)again
3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C)again
4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C)again
5.pcI(SP)+RBS+crtZ+terminator(XP)(zip)(anti-A)again
6/30/2011ligation RBS+ilvC and RBS+ilvD and RBS+malssonly 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) grown
2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) grown
5.pcI(SP)+RBS+crtZ+terminator(XP)(zip)(anti-A) grown
colony PCR above-mentioned
electrophoresis check
sequencing above-mentioned		incubate 37RBS_CrtY_Terminator
7/1/2011transform RBS+malss ; digest PSB1A3 ; ligation and transform B0034+kivdextract 37RBS_CrtY_Terminator digest 37RBS_CrtY_Terminator(X,P)
7/4/2011RBS+malss colony PCR
7/5/2011transform RBS+ilvC and RBS+ilvD ; ligation Ptet+vioABCE
7/6/2011RBS+ilvC and RBS+ilvD colony PCR
7/7/2011Electrophoresis of RBS+ilvC and RBS+ilvD and RBS+malss
7/9/2011sequencing result → fail
digest RBS+crtZ+terminator(XP)(zip)(anti-A)
digest RBS+crtZ+terminator(XP)(purify)(anti-A)
digest pcI(ES&SP)(anti-A)
digest PSB1K3(EP)(anti-K)
digest PSB1C3(EP)(anti-C)
ligate 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)
2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)
3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C)
4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C)
5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A)
7/11/2011Ptet+vioABCE colony PCRtransform 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)
2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)
3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C)
4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C)
5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A)
7/12/2011Electrophoresis of Ptet+vioABCE only 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) grown
2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) grown
5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) grown
7/13/2011Ptet(SP) and vioABCE(XP) gel purified ; ligation Ptet+vioABCE
7/14/2011transform Ptet+vioABCEFlow cytometry: K098988, K098987 (in 25,37,42 temperature degree ) colony PCR 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)
2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)
5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A)
electrophoresis check
sequencing 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)
2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)
5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A)
7/16/2011digest CrtEBI(E,S)
7/17/2011sequencing result 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) → successs
incubate cI+pcI-K098995 in LB-medium
7/18/2011ligation and transform B0034+kivd ; Ptet+vioABCE colony PCRextract plasmid cI+pCIligase CrtEBI+37RBS_CrtY_Terminator
7/19/2011Electrophoresis of Ptet+vioABCE digest cI+pcI(ES)(anti-A)
digest RBS+ctrZ+terminator(XP)(anti-A)
digest PSB1K3(EP)(anti-K)
ligate cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)		Transform CrtEBI+37RBS_CrtY_Terminator
7/20/2011ligation RBS+Alss ; prepare plasmid of Ptet+vioABCE,kivd,ilvC,ilvD,Alss ; B0034+kivd colony PCR ; incubate LB of PSB1A3 and PSB1K3 transform cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)
7/21/2011prepare plasmid of PSB1K3 ; Electrophoresis of B0034+kivd ; transform RBS+Alss
7/25/2011ligation RBS+Alss ; RBS+Alss colony PCRcolony PCR cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)
electrophoresis check
sequencing cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)		ligase CrtEBI+37RBS_CrtY_Terminator
7/26/2011digest B0034+ilvC and B0034+ilvD ; transform RBS+Alss Transform CrtEBI+37RBS_CrtY_Terminator
7/27/2011ligation K098995+B0034 ; RBS+Alss colony PCR ; ligation (B0034+ilvC)+(B0034+ilvD)
7/28/2011 RBS+Alss colony PCR again ; transform K098995+B0034 and (B0034)ilvC+ilvDcolony PCR CrtEBI+37RBS_CrtY_Terminator→success
7/29/2011 (B0034)ilvC+ilvD colony PCR ; ligation (B0034)ilvC+ilvD and K098995+B0034
7/30/2011transform (B0034)ilvC+ilvD and K098995+B0034 ; digest RBS+ilvD(ES)
7/31/2011K098995+B0034 and (B0034)ilvC+ilvD colony PCR
8/1/2011colony PCR of ilvD+terminator and ligation RBS+ilvC+ilvD(PSA1C3)and then transformsequencing result cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) → successs
8/2/2011抽plasmid ptet+vioABCE+terminator and colony PCR RBS+ilvC+RBS+ilvD and incubate37C RBS+tetR+terminatorFlow cytometry: K098988 in 42 temperature degree
8/3/2011ligase (B0030,B0032)ilvC+ilvD and digest B0032+COO12,BOO32+Alss and 抽plasmid Ptet+vioABCE+terminator then digest it and transform 37C RBS+tetR+terminator and (B0030,B0032)ilvC+ilvDligase CrtEBI+Plac CrtEBI+Ptet
8/4/20111.ligaes 2.check digestion product 3.抽plasmid 37C RBS+tetR+terminator 4.transform ligastion pruduct Plac+Alss,RBS+ilvD+37C RBS+tetR+terminator 5.colony PCR ilvC+ilvD1.digest B0015 (XP) and check by electrophoresis 2.ligate B0030 or B0034 +kivd and B0015transform CrtEBI+Plac CrtEBI+Ptet ligase J23103_CrtEBI+37RBS_CrtY_Terminator
8/5/20111.colony PCR RBS+ilvD+37C RBS+tetR+terminator 2.colony PCR Plac+Alss 3.1. transform 8/4 ligation products 2.purify Boo15, J61048, Ptet and check by electrophoresis 3. ligate Ptet(purify) and kivd, J61048(purify) and kivd, B0015(purify) and kivd 4.Flow cytometry Transform J23103_CrtEBI+37RBS_CrtY_Terminator
8/6/20111.ligase RBS+ilvD+37C RBS+tetR+terminator 1.PCR kivd+Boo15 2. Check PCR products by electrophoresis. Four samples reach expected bands. 3. transform 8/5 ligation products 4. inbate 988 DH5-alpha, 988 EPI300 5. four zone culture kivd+B0015colony PCR
8/7/20111.extract plasmid:EPI300 K098988, DH5-alpha K098988 and check py electrophoresis 2.sequencing kivd+J61048, Ptet+kivdpurify CrtEBIY
8/8/20111.transform RBS+ilvD+37C RBS+tetR+terminator 1.transform 988 to EPI300 2.sequencing kivd+B0015
8/9/20111.ligase PCI +B0034 and (B0032)ilvC+ilvD+37C RBS+tetR+terminator and B0032+C0012+R+LuxR+terminator 2.transform (B0032)ilvC+ilvD+37C RBS+tetR+terminator 3.digest B0034 and (B0032)ilvC+ilvD and Plac+B0032+Alss1.digest kivd+J61048, Ptet+kivdligase Plac+CrtEBIY(purify) digest J23103CrtEBI(E,S).(S,P)
8/10/20111.ligate Plac+Alss+ilvC+ilvD 2.digest(B0032)ilvC+IlvD and B0030,32,34+ilvC 3.check digestion product 4.colony PCR RBS+ilvD+37C RBS+tetR+terminator 5.transform (B0030,34)ilvC+ilvD and Plac+Alss+ilvC+ilvD and (B0032)ilvC+IlvD and pCI+B00341.ligate Ptet+kivd+terminator with different methods 2.Flow cytometry: incubate K098988(EPI300) at 32, 37, 42 temperature degreeTransform J23103CrtEBI+37RBS_CrtY_Terminator
8/11/20111.ligate Plac+Alss+RBS+ilvC and ilvC+ilvD+37C RBS+tetR+terminator 2.colony PCR Plac+Alss+ilvC+iulvD and pCI+B0034 and ilvC+ilvD+37C RBS+tetR+terminator 3.transform ligastion products 5.ligate Plac+Alss+ilvC+ilvDligase J23103CrtEBI+37RBS_CrtY_Terminator colony PCR →success
8/12/20111.ligate Plac+Alss+ilvC and iulvC+ilvD+37C RBS+tetR+terminator 2.check digestion products(8/10,11) 3.colony PCR Plac+Alss+ilvC and (B0034)ilvC+ilvD and ilvC+ilvD+37C RBS+tetR+terminator 4.transform Plac+Alss+ilvC+ilvD and ilvC+ilvD+37C RBS+tetR+terminator
8/13/20111.colony PCR 8/12transformation products 2.liagte B0030,34+ilvC+ilvD and Plac+Alss+RBS+ilvC and then transform1.digest plasmids of kivd EcoR1 and Pst1 2.ligate kivd with psb1c3
8/14/20111.digest ilvC+ilvD+37C RBS+tetR+terminator 2.check digestion products 3.ligate Plac+Alss+ilvC+ilvD+37C RBS+tetR+terminator(PSB1K3) 4.digest Plac+Alsstransform 8/13 ligation products
8/15/20111.ligate Plac+Alss+B0030,32,34+ilvC and (B0030,34)ilvC+ilvD 2.抽plasmid Ptet+Alss+ilvC+ilvD 3.incubate LB of transformation products 4.transform Plac+Alss+B0030,32,34+ilvC and Plac+Alss+ilvC+ilvD+37C RBS+tetR+terminator and B0030,34+ilvC+ilvD1.PCR 8/14 sample and check by electrophoresis->fail 2.digest plasmids of crtZ EcoR1 and Pst1 3.ligate crtZ and psb1c3
8/16/20111.抽plasmid of LB products(8/15) 2.digest Plac+Alss+ilvC+ilvDincubate psb3T5
8/17/20111.check dugestion products 2.colony PCR Plac+Alss+ilvC+ilvD+37C RBS+tetR+terminatortransform 8/15 ligation productsextract plasmid psb3T5
8/18/20111.ligate ilvC+ilvD and Plac+Alss+RBS+ilvC and pCI+B0034 2.colony PCR B0034+C0012+37C RBS+tetR+terminator 3.transform ligation productsPCR kivd and check by electrophoresis->successdigest k098995_CrtZ_Terminator(X,P) psb3T5(E,P) ligase 1.J23103CrtEBIY+K098995_CrtZ_Terminator+psb1c3/psb3T5 2.J23103CrtEBIY+psb1c3/psb3T5
8/19/20111.colony PCR 8/18 transformation products 2.transform Plac+Alss+RBS+ilvC and pCI+B0034PCR crtZ and check by electrophoresis->successtransform 1.J23103CrtEBIY+K098995_CrtZ_Terminator+psb1c3/psb3T5 2.J23103CrtEBIY+psb1c3/psb3T5
8/20/2011incubate kivd in LBcolony PCR
8/21/2011
8/22/20111.colony PCR 8/19 transformation products 1.ligate Ptet+kivd+terminator+psb1c3 2.transform 3.incubate crtZ in LB 4.extract plasmid of kivd and check by electrophoresisextract plasmid J23103_CrtEBIY digest J23103_CrtEBIY(E,P) and check by electrophoresis ligase J23103_CrtEBIY+K098995_CrtZ_Terminator
8/23/20111.digest Plac+Alss+ilvC+ilvD 2.ligase(Plac+Alss+ilvC+ilvD)and(B0032+ilvC+B0032+ilvD) + (37C RBS+tetR+terminator) 3.colony PCR 8/18 pCI+B0034 4.transform ligation products 1.extract plasmid of crtZ and check by electrophoresisdigest 1.psb3T5(E,P) 2.psb1c3(E,P) 3.J23103_CrtEBIY(E,S) 4.K098995_CrtZ_Terminator(E,X) 5.CrtY(E,P) 6.CrtY_Terminator(E,P) purify 1.psb3T5(E,P) 2.psb1c3(E,P) 3.J23103_CrtEBIY(E,S) 4.K098995_CrtZ_Terminator(E,X) ligase 1.CrtY+psb1c3 2.CrtY_Terminator+psb1c3 transform J23103_CrtEBIY+K098995_CrtZ_Terminator
8/24/20111.colony PCR Plac+Alss+ilvC 2.ligase and transform Plaac+Alss+ilvC+ilvD+37C RBS +tetR+terminator ,(B0034)ilvC+ilvD 3.incubate LB of B0034+C0012+37C RBS+tetR+terminator 1.ligate PcI+rbs+crtZ+T and psb1c3 2.PCR Ptet+kivd+Tcolony PCR J23103_CrtEBIY+K098995_CrtZ_Terminator transform 1.CrtY+psb1c3 2.CrtY_Terminator+psb1c3
8/25/20111.colony PCR 8/24 transformation products 2.ligase Plac+Alss+B0032,34+ilvC ,(B0032)ilvC+ilvD+37C RBS+tetR+terminator 3.抽plasmid of 8/24 LBtransform PcI+crtZ+terminatorcolony PCR 1.CrtY+psb1c3 2.CrtY_Terminator+psb1c3 →success
8/26/20111.transform 8/25 ligation products 2.digest K098995,37C RBS+tetR+terminator,(B0030)ilvC+ilvD 3.ligase K098995+B0034
8/27/20111.colony PCR BOO32+ilvC+ilvD+37C RBS+tetR+terminator ,(B0034)ilvC+ilvD 2. transform 8/26 ligation products
8/28/20111. colony PCR 8/27 transform products and K098995+B0034
8/29/2011digest psb4A5
8/30/20111.digest Ptet+kivd+T EcoR1 and Pst1 2. transform Ptet+kivd+T+psb4A5
8/31/20111.digest PSB1C3 2.ligase Plac+Alss+ilvC+ilvD ,(B0034)ilvC+ilvD ,37C RBS+tetR+terminator ,B0034+ilvC ,Plac+Alss+B0030+ilvC ,B0030+ilvD+37C RBS+tetR+terminator 3.transform Plac+Alss+B0032,34+ilvC1.digest 37RBS+tetR+T,Ptet+kivd+T 2. transform Ptet+kivd+T+psb4A5 3.digest Plac+Alss+ilvc+ilvd (SP) and 37RBS+tetR+T(XP)
9/1/2011in morning :1.ligation for change parts to 1c3 backbone 2.PCR pLac+Alss+Boo32+ilvC and check by electrophoresis //in afternoon: 3.transform ligation product in the morning for change parts to 1C3 backbone parts are a. b0030+ilvc b.b0034+ilvc c. b0030+ilvD d. b0032+ilvd e. Plac +Alss+ilvc+ilvD f.b0034+lacI +37℃RBS+tetR+T g. B circuit 6-1 7-1 4. PCR 8/31 product and check by electrophoresis // 5.select&culture PSB1A3 and Dcircuit inLB 1.ligation 37RBS+tetR+T with Ptet+kivd+T and transform they 2.PCR 8/30 sample3.purify and ligate Plac+Alss+ilvc+ilvd (SP) and 37RBS+tetR+T(XP) 4.transform Plac+Alss+ilvc+ilvd + 37RBS+tetR+T digest 1.J23103_CrtEBIY(E,S) 2.K098995_CrtZ_Terminator(E,P) 3. psb4A5(E,P)
9/2/2011in morning : 1. extract plasmid of LB cultured last night //in afternoon: 2. PCR 9/1 change backbone product and check by electrophoresis 3. transform 9/1 product totally 16 tubesligase K098995_CrtZ_Terminator+psb4A5
9/3/20111.PCR 9/2 product & check by electrophoresis => success parts:pLac+alss+B0030+ilvC //B0034+ilvC+ilvD 2.culture right partsPCR 37RBS+tetR+T+Ptet+kivd+T and sequence one of the samplestransform K098995_CrtZ_Terminator+psb4A5
9/4/20111.ligation D circuit to PSB1C3&PSB3K3 for change backbone 2.ligation B circuit & B0032+ilvD &(B0030)pLac+alss+ilvc+ilvd to PSB1C3 for change backbone 3. ligation a. (pLac +alss) with (B0032/34+ilvc) b.(B0034+ilvc) with (B0034+ilvD) colony PCR K098995_CrtZ_Terminator+psb4A5 →success
9/5/20111. PCR 9/3 product & check by electrophoresis
9/6/2011in morning :1.transform 9/4 ligation product //in afternoon:1. select bacteria and delivery sequencingextract plasmid K098995_CrtZ_Terminator+psb4A5
9/7/2011cotransform J23103_CrtEBIY_psb1k3+K098995_CrtZ_Terminator_psb4A5
9/8/20111.PCR 9/6 transformation product incubate cotransform product in different temperature
9/9/20111.check 9/8 PCR product by electrophoresis
9/10/2011digest (K098995+b0034) &vioC
9/14/20111. prepare for spreading the incubated LB on plates &incubated in 37 ℃ 2. ligation ptet +vioABE +PSB1c3 3. digest (K098995+B0034) E.P&E.S and PSB1c3 4.ligation parts for change backbone to 1C3 :a. B circuit 7-1 7-2 b.B0030+ilvD c. B0032+ilvD d. B0030+ilvC e. B0034+ilvC f. D circuit -2&-3cotransform J23103_CrtEBIY_psb3T5+K098995_CrtZ_Terminator_psb4A5
9/15/2011in morning :1. digest PSB1A3 2. transform PSB1A3 &(vioABE + PSB1C3) 3. incubated colony to show different color in different temperature 4. inactive vioC+T digest product //in afternoon : 1. ligation(K098995 +B0034) with (vioC) 2. transform product for change backbone //at night: incbated D circuit in LBincubate cotransform product
9/16/20111. PCR 9/15 transform product &check by electrophoresis 2. PCR vioABE+1C3// night: 3.transform PSB1A3 &(K098995+B0034+vioC+T) 4. ligation the other failed parts for change backbone to 1C3 5. extract plasmid of D circuit and check by electrophoresis 6. incubated (B0034+ilvC) (B0030+ilvD) (B circuit 7-1) (K098995 +B0034) (vioABE )=>1C3 inLB in37℃spread the incubated LB on plates and incubate in different temperature
9/17/20111. transform 9/16 product 2. religation PSB1A3 &(K098995+B0034+vioC+T)
9/18/20111.transform 9/17 product 2.PCR (B0032+alss) (B0030+ilvD) (B0030+ilvC) &check by electrophoresis
9/19/20111. incubated parts in LB 2. digest 9/16 plasmid to check 3. ligation C circuit Flow cytometry: 2010 IGEM
9/20/20111. transform C circuit 2. check (D circuit -1/-2)(B0034+ilvc-1/-2)(B0030+ilvd-1/-2)(K098995+B0034-1/-2)(B circuit-1/-2)(PSB1A3 E.P) by electrophoresis 3. extract plasmid of (B0030+ilvD)(B0032+ALss) and digest it
9/21/20111.digest K098995+B0034 2. check 9/20 digest product by electrophoresis
9/22/2011digest Plac+alss+ilvc+ilvd (EP) and ligate is with psb3K3
9/23/20111.transform Plac+alss+ilvc+ilvd in the morning 2.incubate Plac+alss+ilvc+ilvd in LB at night
9/24/20111.extract plasmid of Plac+alss+ilvc+ilvd 2.cotransform Plac+alss+ilvc+ilvd(3K3) and Ptet+kivd+T(4A5)
9/27/2011incubate cotransform product in different temperature

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