"
Lab Notebook
5/21/12 week
-Megan transformed casBCED12 with Amp resistance, casBCED12 with Cm resistance, pSBIC3_RFP, pSBIK3_RFP, and pSBIA3_RFP
-successful results: pSBIC3_RFP and pSBIK3_RFP.
-Luke inoculated pSBIC3_RFP and pSBIK3_RFP
-Stephan miniprepped pSBIK3_RFP, and pSBIT3_RFP
-all very low concentration
-Ellen miniprepped pSBIC3_RFP, pSBIA3_RFP
-concentration (got 98 and 120 ug/ml, respectively)
-Rachel digested pSBIC3 from pSBIC3_RFP, pSBIK3 from pSBIK3_RFP
-Transformed pSBIT3_RFP, pSBIA3_RFP, casBCDE12 on Amp
-Digested casABCDE123, tetO-GFP
-Ligated casA,3 with tetO-GFP, with pSBIA3 or pSBIC3
-Ligated casBCDE12 with pSBIA3 or PSBIC3
5/28/12 week
-Inoculated (2x) 5mL psB1A3_RFP and psB1T3_RFP.
-Transformed psB1C3_RFP, tetO_GFP, and psB3T5_RFP.
-Miniprepped psB1C3_RFP and psB1K3_RFP
-Transformed psB1C3-RFP, psB3T5-RFP, tetO-GFP
-PCRed fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK
-Gel extracted and digested the genes with EP or EX
-Miniprepped pSB3T5_RFP (3) and tetO-GFP
-Digested tetO-GFP 2
6/4/12 week
-PCRed genes using BL21 and HT115
-Gel extracted and digested
-Ligated the genes to corresponding psb1C3_RFP vectors
-Transformed 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A
-successful: 4-12, 3-8D, 3-2H, 1-9F, 3-2J. (very low efficiency)
-Transformed cheB, cheC, cheY, cheZ, luxS, lsrK, lsrR, flaU, flbA, flhB, flhD, flgJ, flgK, fliA, fliC, fliD, fliF, fliG, fliH, fliM
-Inoculated 4-12A, 3-8D, 3-2H, 1-9F, 3-2J
-Inoculated HT115 (w/ 5uL tet) and HB101 (w/o antibiotics)
-PCRed fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK
6/11/12 week
-Digested fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK
-Gel extracted fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK
-PCRed fliC with DH5alpha, BL21, HT115
-Ligated cm1, cm2, rfp1, rfp2
-Gel purified cm1, cm2, rfp1, rfp2
-Transformed fliA, cheZ, cheY, fliH, fliG, flgJ, flihB, flgK, fliD,fliM, 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A
-successful: 1-9F, 1-14H, 1-18H, 3-2H, 3-2J, 3-8D, 4-12A
-Digested cm vector with EP
-Gel extracted cm vectors
-Inoculated 3-8D, 1-9F, 4-12A
-PCRed chA, cheB, cheC, cheY, cheZ
-Transformed ligation products: fliG, flgJ, cheY, cheZ, fliD, flhB, fliM, fliH, flgK
-Ligated fliM, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2 to Cm vector, fliH
-Miniprepped the innoculations (3-8D, 1-9F, 4-12A)
-low concentrations
-Transformed fliH, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2, cm vector
-Miniprepped 1-9F(1), 1-9F(2), 3-8D(1), 3-8D(2), 4-12A(1), 4-12A(2)
-low concentrations
6/18/12 week
-PCRed lsrR(BL21), lsrR(HT115), lrsK(BL21), lsrK(HT115)
-Inoculated 1-9F, 3-8D, 4-12A
-Miniprepped 1-9F, 3-8D, 4-12A
-low concentrations
-Transformed J23100, RBS B0034, luxS, GFP E0040
-successful
-Inoculated J23100, RBS B0034, luxS, GFP E0040
-PCRed cheA, cheB, cheY, cheZ using HTB genomic DNA
-PCRed fliC(1), motA(2), motB(3) using DH5alpha
-Miniprepped 1-9F, 3-8D, 4-12A
-acceptable concentrations
-Digested cheA, cheB, cheY, cheZ and gel purified
-Ligated cheA, cheB, cheY, cheZ
-Transformed cheA, cheB, cheY, cheZ
-successful
-Digested psB1C3 with X and X/S
-Digested fliC, motA, motB
-Miniprepped J23100, GFP E0040, luxS, RBS Boo34
-low concentrations
6/25/12 week
-Transformed motA, motB, and control
-Inoculated T7 promoter and cheY
-Colony PCRed 20 colonies from motA, motB
-Inoculated motA, motB, cheA, cheB, cheY, cheZ
-Miniprepped cheZ, cheY, cheA, cheB, motA, motB
-acceptable concentrations
-Glycerol stock: motA 6-14, cheZ, cheY, cheA, cheB, motA, motB
-Transformed cheZ, pLaeI+RBS+cheZ, RBS+cheZ+dbl terminator, cheZ w/ H13 tag+dbl terminator, cheZ w/ H13 tag, strong promoter w/RBS
-Inoculated cheZ, pLaeI+RBS+cheZ, RBS+cheZ+dbl terminator, cheZ w/ H13 tag+dbl terminator, cheZ w/ H13 tag, strong promoter w/RBS
-PCR miniprep of cheA(7,16), cheB(14), cheZ(14), motA(15)
7/2/12 week
-Miniprepped cheZ w/ histag, cheZ, cheZ w/ RBS + dbl termination, pLacI w/ RBS + cheZ, cheZ w/ Histag + dbl termination, strong promoter w/ RBS (cm)
-acceptable concentrations
-Inoculated cheZ w/ histag (amp), cheZ (amp), cheZ w/ RBS, double termination (amp), pLacI w/ RBS, cheZ (amp), cheZ w/ histag, double termination (amp), strong promoter w/ RBS (cm)
-Miniprepped: psB1C3RFP (5 times), cheZ, strong promoter+RBS+cm, RBS+cheZ+dbl term site, pLacI+RBS+cheZ, cheZ+Hist+dbl, cheZ+Histag
-acceptable concentrations
7/9/12 week
-Digested and gel extracted fliC and GFP
-Ligated fliC
-Transformed fliC-GFP
-Received and plated strains: BW30044, BW30045, K-12 wildtype strain, strain B wildtype, JW1871-1, JW1879-2, JW2662-1, MG1655 (motile), W3110
7/16/12 week
-Made competent cells MG1655
-digested GFP vector for control using XbaI
-Colony PCRed the fliC GFP
-primer dimers. unsuccessful-
7/23/12 week
-PCRed fliC w/ RBS
-Transformed GFP-DH5a, GFP-MG1655
-Inoculated w/ 50uL transformation product
-Plated GFP-DH5a, GFP-MG1655
-PCRed fliC w/ RBS
-Miniprepped GFP-DH5a (x2), GFP-MG1655 (x2)
-acceptable concentrations
-Digested fliC and GFP w/ XbaI and gel purified
-Ligated fliC-GFP and GFP w/ H2O (control)
-Transformed 1hour ligation product
-unsuccessul
-Made control and cheY mutagenesis solutions
-Transformed fliC-GFP and GFP w/ H2O control into DH5a cells
-successful: GFP + H2O control and fliC-GFP
-Quickchange mutagenesis
-Colony PCR fliC-GFP
7/30/12 week
-Miniprepped fliC-GFP
-acceptable concentrations
-Digested miniprepped products
-Quickchange PCRed fliC-GFP
-Digested quickchange PCR products with DPN1
-Digested fliC minipreps with Spe1
-successful
-Transformed 43, 44, quickchange digests
8/6/12 week
-Digested cheY
-Quickchange PCRed fliC and cheY with control
-Digested fliC-GFP, cheY, control
-Plated fliC, cheY, and pUC18
-Inoculated cheY mut colonies
-Miniprepped cheY mut
-Glycerol stocks of cheY mut
-PCRed cheY mut
-Digested cheY mut
8/13/12 week
-PCRed fliA, fliD, fliE, fliF, fliG, fliH, fliM, flhB, flhC, flhD, flgA, flgB, flgJ, flgK, fliC-GFP, lsrR, lsrK
-Digested and gel extracted the flagella genes
-Transformed fliA, fliD,fliF, fliM, flhB, flgB, flgJ, flhD, lsrR, lsrK
-Digested fliC-GFP with DpnI, BBa_K318512, BBa_K338001, BBa_K216005
-Transformed pSB1C3 and fliC-GFP mut
-Inoculated pSB1C3-RFP, BBa_K318512, BBa_K338001, BBa_K216005
-Miniprepped pSB1C3 and BBa_K318512, BBa_K338001, BBa_K216005
-acceptable concentrations
-Digested pSB1C3 fliF, fliD, flhB, flhD, flgJ, fliM, fliA
-Ligated fliF, fliD, flhB, flhD, flgJ, fliM, fliA with pSB1C3
8/20/12 week
-Colony PCRed fliA, fliM, fliD, fliF, flgJ, flhB, flhD
-Imaged MG1655 in various conditions: pH 5, 6, 8, 9; NaCl 50mM, 100mM, 200mM, 400mM, 500mM; temperature 15C, 20C, 42C; glucose 10mM, 20mM, 50mM
-Miniprepped fliC-GFP mut, cheY mut
-acceptable concentrations
-Glycerol stocks of fliC-GFP, cheY
-Colony PCRed flgJ, flhD, fliA, flhB, cheY
-Inoculated successful colonies
-Colony PCRed fliM, fliF, fliD
-Glycerol stocks of fliA, flhB, flgJ, flhD
-Imaging of MG1655
-Miniprepped fliA, flhB, flgJ, flhD
-Colony PCRed fliM, fliD, fliF
-Inoculated fliM
-PCRed flhD, flhB, flgJ, fliA to test directionality
8/27/12 week
-PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR
-Digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR
-all successful except ompR, metR, araC
-Digested pSB1C3
-Ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter to pSB1C3
9/3/12 week
-Digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns
-Transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406
-Digested pSB1C3
-Ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns with pSB1C3
9/10/12 week
Transformed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns
-Inoculated transformations
-Colony PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns
-Transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406
-Colony PCRed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406
-Transformed BBa_K398108, BBa_K398406
-Inoculated BBa_K398108, BBa_K398406, fliH
-Miniprepped BBa_K398108, BBa_K398406, fliH
-acceptable concentrations
-Colony PCRed BBa_K398108, BBa_K398406, fliH
-Miniprepped flhD
9/17/12 week
-Transformed arabinose
Inoculated cheY, T7
-Digested cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC
-Ligated cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC
-Miniprepped T7, cheY
-acceptable concentrations
-Transformed T7/p.cola, pACYC
-Colony PCRed cheZ RBS, cheZ T7, p.cola cheY
-Inoculated cheZ RBS, cheZ T7, p.cola cheY
-Miniprepped cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
-Transformed cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
-Inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
-Imaged cheZ, cheZ T7, cheZ RBS T7, cheZ T7
-Glycerol stocks of cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
-Imaging of pcola cheZ A, cheZ RBS T7, cheZ RBS T7, cheZ T7, pcdf cheY A
-Inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
9/24/12 week
-Imaged BBa_K398108, cheZ RBS T7, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
-Imaged cheZ RBS T7, cheZ T7, pcdf cheY, control
-Digested cheY p.cola, pcdf cheY, pcdf cheZ, pcola cheZ, cheZ T7 RBS
-Transformed T7 RBS cheZ
-Inoculated salt biobricks from frozen stocks
-Colony PCRed T7 RBS cheZ
-Miniprepped cheZ RBS T7
10/1/12 week
-Miniprepped cheA, cheB, cheY, cheZ, motA. motB, T7 RBS cheZ, T7 RBS cheY, flgJ, fliA, fliH, fliC-GFP, flhB, flhD
-Western blot for T7 RBS cheY, T7 RBS cheZ
