Team:Austin Texas/Week of July 9 to July 15

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= Notebook for week of July 9 to July 15 =

== Design of Converter Plasmid ==

*Razan 
**7/9- Gibson Assembly of Converter and Control plasmids
**7/10- Transformation of gibson failed and gel shows no correct bands
**7/11- Redid Gibson assembly of Converter and Control plasmids with longer incubation time; same results.

== Design of Reporter Plasmid ==

* Ben
** Made 5 uM stocks of all pReporter primers
** '''PCR:'''
*** pIGM023 (pET21.KOD-BB): JE.pET.BB.F, JE.pET.BB.R
*** pIGM022 (pAK.TAQ-rrnB): JE.rrnB.F, JE.rrnB.R
*** pIGM006 (pSB1A2-K081009 [LasI]): JE.LasI.F, JE.LasI.R
*** pIGM005 (pSB1AK3-K081014 [RFP]): BS.RFP.F, BS.RFP.R
*** pIGM025 (pACYC.T7.GFP-sfGFP): BS.sfGFP.F, JE.sfGFP.R  '''*Note*: used incorrect template. Will use pIGM026.'''
*** pIGM008 (pSB1AK3-B0015 [double.terminator]): JE.GFPTerm.F, JE.GFPTerm.R
*** '''Results:''' No prepped plasmids available as template for IGM005-6, so I tried using BioBrick transforming DNA as template; these rxns didn't work. Will try again with prepped DNA.
** Made test tube cultures with  IGM004 (pSB3K3-I714891 [GFP]), IGM005 (pSB1AK3-K081014 [RFP]), and IGM006 (pSB1A2-K081009 [LasI]) for the retry.
** Prepped DNA.
** '''PCR:''' (retry of previous with new templates)
*** pIGM023 (pET21.KOD-BB): JE.pET.BB.F, JE.pET.BB.R
*** pIGM022 (pAK.TAQ-rrnB): JE.rrnB.F, JE.rrnB.R
*** pIGM006 (pSB1A2-K081009 [LasI]): JE.LasI.F, JE.LasI.R
*** pIGM005 (pSB1AK3-K081014 [RFP]): BS.RFP.F, BS.RFP.R
*** pIGM026 (pET21-sfGFP): BS.sfGFP.F, JE.sfGFP.R 
*** pIGM008 (pSB1AK3-B0015 [double.terminator]): JE.GFPTerm.F, JE.GFPTerm.R
*** '''Results:''' All lanes contained correct-size bands.
** Cleaned up DNA.
** '''OE-PCR:'''
*** BS.LoxLac.F1 + BS.LoxLac.F2 + BS.LoxLac.R1
*** BS.LoxLacFlipped.F1 + BS.LoxLacFlipped.F2 + BS.LoxLacFlipped.R1
*** '''Results:''' Accidentally used 10X excess insert instead of 10X excess of outer primers. Will retry with correct conditions.
** '''OE-PCR:'''
*** BS.LoxLac.F1 + BS.LoxLac.F2 + BS.LoxLac.R1
*** BS.LoxLacFlipped.F1 + BS.LoxLacFlipped.F2 + BS.LoxLacFlipped.R1
*** '''Results:''' All lanes contained correct-size bands.
** Gel-extracted LoxLac and LoxLacFlipped sequences. 
** '''PCR:'''
*** LoxLac: BS.LoxLac.F1, BS.LoxLac.R1
*** LoxLacFlipped: BS.LoxLacFlipped.F1, BS.LoxLacFlipped.R1
*** '''Results:''' All lanes contained correct-size bands.
** Cleaned up DNA.
** '''OE-PCR:'''
*** rrnB + LasI: JE.rrnB.F, JE.LasI.R
*** RFP + LoxLac: BS.RFP.F, BS.LoxLac.R1
*** RFP + LoxLacFlipped: BS.RFP.F, BS.LoxLacFlipped.R1
*** sfGFP + double.terminator: BS.sfGFP.F, JE.sfGFP.R
*** '''Results:''' All lanes contained correct-size bands except for RFP + LoxLacFlipped. Will retry this rxn.
** '''OE-PCR:'''
*** RFP + LoxLacFlipped: BS.RFP.F, BS.LoxLacFlipped.R1
*** '''Results:''' All lanes contained correct-size bands.
** '''PCR:'''
*** rrnB.LasI
*** RFP.LoxLac
*** RFP.LoxLacFlipped
*** sfGFP.double.terminator
*** '''Results:''' All lanes contained correct-size bands.
** Cleaned up DNA, ran on gel, and gel extracted.
** Dpn1 digested pET21.BB amplicon. Cleaned up.
** '''Gibson assembly:'''
*** pET21.BB (control)
*** pET21.BB + rrnB.LasI + RFP.LoxLac + sfGFP.double.terminator
*** pET21.BB + rrnB.LasI + RFP.LoxLacFlipped + sfGFP.double.terminator
*** '''Results:''' No visible product band.
** Desalted, transformed, and plated on LB+Cab.

== Spinach Aptamer ==

== CBB5 Caffeine Inducible Promoters ==
*Peter
**7/9 - Set up confirmation PCR of cultures. 1 for each culture, as well as 2 controls from CBB5 genomic DNA.
**7/10 - Gel electrophoresis of confirmation PCR showed 2 successful Gibson product colonies, as well as both successful controls from CBB5 genomic DNA.
**7/11 - Miniprep of ORF1 - 1 and -2. Submit minipreped DNA for sequencing
**7/12 - Sequencing failed
**7/13 - Resubmitted sequencing with new primer stocks.

== Inducible Odor ==