iGEM 2012 — Team USC

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# Team:USC/Notebook

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Lab Notebook « E. musici

* [E. Musici](https://2012.igem.org/Team%3AUSC)
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[E. musici](https://2012.igem.org/Team%3AUSC "E. musici")

# [E. musici](http://uscigem2012.wordpress.com/)

University of Southern California iGEM 2012

## 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)[![](http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg?w=442&h=292 "DSC_0028")](http://uscigem2012.files.wordpress.com/2012/07/dsc_0028.jpg)

-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

[![](http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg?w=412&h=274 "_MG_4058")](http://uscigem2012.files.wordpress.com/2012/07/mg_4058.jpg)**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[![](http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg?w=512&h=338 "DSC_0026")](http://uscigem2012.files.wordpress.com/2012/07/dsc_0026.jpg)
 -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[![](http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg?w=496&h=331 "_MG_4057")](http://uscigem2012.files.wordpress.com/2012/07/mg_4057.jpg)

-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*

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