Team:USC/Notebook

From 2012.igem.org

Lab Notebook « E. musici

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
-Eric transformed pSBIT3_RFP, pSBIA3_RFP, casBCDE12 on Amp
-Stephan digested casABCDE123, tetO-GFP
-Stephan digated casA,3 with tetO-GFP, with pSBIA3 or pSBIC3
-Eric digated casBCDE12 with pSBIA3 or PSBIC3

5/28/12 week
-Megan inoculated (2x) 5mL psB1A3_RFP and psB1T3_RFP.
-Rebecca transformed psB1C3_RFP, tetO_GFP, and psB3T5_RFP.
-Rebecca miniprepped psB1C3_RFP and psB1K3_RFP
-Ellen transformed psB1C3-RFP, psB3T5-RFP, tetO-GFP
-Rachel PCRed fliE, fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK
-Luke gel extracted and digested the genes with EP or EX
-Megan miniprepped pSB3T5_RFP (3) and tetO-GFP
-Rachel digested tetO-GFP 2

6/4/12 week
-Stephan PCRed genes using BL21 and HT115
-Stephan gel extracted and digested
-Ellen digested the genes to corresponding psb1C3_RFP vectors
-Rebecca 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)
-Rachel transformed cheB, cheC, cheY, cheZ, luxS, lsrK, lsrR, flaU, flbA, flhB, flhD, flgJ, flgK, fliA, fliC, fliD, fliF, fliG, fliH, fliM
-Rachel inoculated 4-12A, 3-8D, 3-2H, 1-9F, 3-2J
-Luke inoculated HT115 (w/ 5uL tet) and HB101 (w/o antibiotics)
-Stephan 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
-Ellen digested fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK
-Luke gel extracted fliM, flhD, flgA, fliD, flhB, fliA, fliE,fliF, fliG, fliH, flgJ, flgK, fliC, cheA, cheB, cheC, cheY, cheZ, luxS, lsrR, lsrK
-Rebecca PCRed fliC with DH5alpha, BL21, HT115
-Rachel ligated cm1, cm2, rfp1, rfp2
-Eric gel purified cm1, cm2, rfp1, rfp2
-Megan 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
-Stephan digested cm vector with EP
-Ellen gel extracted cm vectors
-Ellen inoculated 3-8D, 1-9F, 4-12A
-Eric PCRed chA, cheB, cheC, cheY, cheZ
-Luke transformed ligation products: fliG, flgJ, cheY, cheZ, fliD, flhB, fliM, fliH, flgK
-Rachel ligated fliM, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2 to Cm vector, fliH
-Megan miniprepped the innoculations (3-8D, 1-9F, 4-12A)
-low concentrations
-Rebecca transformed fliH, fliD, flhB, flgK, fliA, cheY, cheZ, fliG, flgJ, rfp1, rfp2, cm vector
-Megan 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
-Rachel PCRed lsrR(BL21), lsrR(HT115), lrsK(BL21), lsrK(HT115)
-Stephan inoculated 1-9F, 3-8D, 4-12A
-Ellen miniprepped 1-9F, 3-8D, 4-12A
-low concentrations
-Luke transformed J23100, RBS B0034, luxS, GFP E0040
-successful
-Stephan inoculated J23100, RBS B0034, luxS, GFP E0040
-Stephan PCRed cheA, cheB, cheY, cheZ using HTB genomic DNA
-Stephan PCRed fliC(1), motA(2), motB(3) using DH5alpha
-Luke miniprepped 1-9F, 3-8D, 4-12A
-acceptable concentrations
-Megan digested cheA, cheB, cheY, cheZ and gel purified
-Luke ligated cheA, cheB, cheY, cheZ
-Megan transformed cheA, cheB, cheY, cheZ
-successful
-Eric digested psB1C3 with X and X/S
-Rachel digested fliC, motA, motB
-Rachel miniprepped J23100, GFP E0040, luxS, RBS Boo34
-low concentrations

6/25/12 week
-Ellen transformed motA, motB, and control
-Rebecca inoculated T7 promoter and cheY
-Rebecca colony PCRed 20 colonies from motA, motB
-Stephan inoculated motA, motB, cheA, cheB, cheY, cheZ
-Luke miniprepped cheZ, cheY, cheA, cheB, motA, motB
-acceptable concentrations
-Rachel glycerol stock: motA 6-14, cheZ, cheY, cheA, cheB, motA, motB
-Luke transformed cheZ, pLaeI+RBS+cheZ, RBS+cheZ+dbl terminator, cheZ w/ H13 tag+dbl terminator, cheZ w/ H13 tag, strong promoter w/RBS
-Rebecca inoculated cheZ, pLaeI+RBS+cheZ, RBS+cheZ+dbl terminator, cheZ w/ H13 tag+dbl terminator, cheZ w/ H13 tag, strong promoter w/RBS
-Stephan PCR miniprep of cheA(7,16), cheB(14), cheZ(14), motA(15)

7/2/12 week
-Megan 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
-Megan 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)
-Ellen 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
-Ellen digested and gel extracted fliC and GFP
-Rachel ligated fliC
-Luke transformed fliC-GFP
-Rebecca 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
-Rachel made competent cells MG1655
-Ellen digested GFP vector for control using XbaI
-Luke colony PCRed the fliC GFP
-primer dimers. unsuccessful-

7/23/12 week
-Ellen PCRed fliC w/ RBS
-Stephan transformed GFP-DH5a, GFP-MG1655
-Megan inoculated w/ 50uL transformation product
-Stephan plated GFP-DH5a, GFP-MG1655
-Rachel PCRed fliC w/ RBS
-Ellen miniprepped GFP-DH5a (x2), GFP-MG1655 (x2)

-acceptable concentrations

-Digested fliC and GFP w/ XbaI and gel purified
-Eric ligated fliC-GFP and GFP w/ H2O (control)
-Eric transformed 1hour ligation product
-unsuccessul
-Stephan made control and cheY mutagenesis solutions
-Megan transformed fliC-GFP and GFP w/ H2O control into DH5a cells
-successful: GFP + H2O control and fliC-GFP
-Luke quickchange mutagenesis
-Rebecca colony PCR fliC-GFP

7/30/12 week
-Megan miniprepped fliC-GFP
-acceptable concentrations
-Rachel digested miniprepped products
-Eric quickchange PCRed fliC-GFP
-Stephan digested quickchange PCR products with DPN1
-Megan digested fliC minipreps with Spe1
-successful
-Megan transformed 43, 44, quickchange digests

8/6/12 week
-Rebecca digested cheY
-Rebecca quickchange PCRed fliC and cheY with control
-Rebecca digested fliC-GFP, cheY, control
-Stephan plated fliC, cheY, and pUC18
-Rachel inoculated cheY mut colonies
-Ellen miniprepped cheY mut
-Stephan glycerol stocks of cheY mut
-Luke PCRed cheY mut
-Luke 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
-Ellen digested and gel extracted the flagella genes
-Stephan transformed fliA, fliD,fliF, fliM, flhB, flgB, flgJ, flhD, lsrR, lsrK
-Megan digested fliC-GFP with DpnI, BBa_K318512, BBa_K338001, BBa_K216005
-Rebecca transformed pSB1C3 and fliC-GFP mut
-Stephan inoculated pSB1C3-RFP, BBa_K318512, BBa_K338001, BBa_K216005
-Ellen miniprepped pSB1C3 and BBa_K318512, BBa_K338001, BBa_K216005
-acceptable concentrations
-Luke digested pSB1C3 fliF, fliD, flhB, flhD, flgJ, fliM, fliA
-Eric ligated fliF, fliD, flhB, flhD, flgJ, fliM, fliA with pSB1C3

8/20/12 week
-Megan colony PCRed fliA, fliM, fliD, fliF, flgJ, flhB, flhD
-Rachel imaged MG1655 in various conditions: pH 5, 6, 8, 9; NaCl 50mM, 100mM, 200mM, 400mM, 500mM; temperature 15C, 20C, 42C; glucose 10mM, 20mM, 50mM
-Rachel miniprepped fliC-GFP mut, cheY mut
-acceptable concentrations
-Stephan glycerol stocks of fliC-GFP, cheY
-Megan colony PCRed flgJ, flhD, fliA, flhB, cheY
-Megan inoculated successful colonies
-Rachel colony PCRed fliM, fliF, fliD
-Rebecca glycerol stocks of fliA, flhB, flgJ, flhD
-Eric imaging of MG1655
-Luke miniprepped fliA, flhB, flgJ, flhD
-Luke colony PCRed fliM, fliD, fliF
-Rebecca inoculated fliM
-Stephan PCRed flhD, flhB, flgJ, fliA to test directionality

8/27/12 week
-Ellen PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns, qseBC promoter, metR, araC, ompR
-Eric 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
-Stephan digested pSB1C3
-Megan 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
-Rachel digested fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns
-Eric transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406
-Stephan digested pSB1C3
-Luke ligated fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns with pSB1C3

9/10/12 week
-Megan transformed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns
-Stephan inoculated transformations
-Rebecca colony PCRed fliA, fliD, fliE, fliG, fliH, fliM, flhC, flhD, qseB, qseC, qseBC, flgA, flgB, flgJ, flgK, hns
-Ellen transformed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406
-Megan colony PCRed BBa_K398108, BBa_J45199, BBa_K398014, BBa_J23039, BBa_K398406
-Rachel transformed BBa_K398108, BBa_K398406
-Luke inoculated BBa_K398108, BBa_K398406, fliH
-Eric miniprepped BBa_K398108, BBa_K398406, fliH

-acceptable concentrations

-Eric colony PCRed BBa_K398108, BBa_K398406, fliH
-Rachel miniprepped flhD

9/17/12 week
-Stephan transformed arabinose
-Stephan inoculated cheY, T7
-Rachel digested cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC
-Luke ligated cheZ, cheZ RBS, cheZ, T7 Stp, cheY, 4-12A Stp, P.cola, pACYC
-Megan miniprepped T7, cheY
-acceptable concentrations
-Rebecca transformed T7/p.cola, pACYC
-Rebecca colony PCRed cheZ RBS, cheZ T7, p.cola cheY
-Rebecca inoculated cheZ RBS, cheZ T7, p.cola cheY
-Stephan miniprepped cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
-Megan transformed cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
-Sean inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
-Megan imaged cheZ, cheZ T7, cheZ RBS T7, cheZ T7
-Ellen glycerol stocks of cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
-Eric imaging of pcola cheZ A, cheZ RBS T7, cheZ RBS T7, cheZ T7, pcdf cheY A
-Luke inoculated cheZ RBS, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ

9/24/12 week
-Rebecca imaged BBa_K398108, cheZ RBS T7, cheZ T7, p.cola cheY, p.cola cheZ, pcdf cheY, pcdf cheZ
-Stephan imaged cheZ RBS T7, cheZ T7, pcdf cheY, control
-Luke digested cheY p.cola, pcdf cheY, pcdf cheZ, pcola cheZ, cheZ T7 RBS
-Megan transformed T7 RBS cheZ
-Rebecca inoculated salt biobricks from frozen stocks
-Rachel colony PCRed T7 RBS cheZ
-Ellen miniprepped cheZ RBS T7

10/1/12 week

-Eric 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