From 2007.igem.org

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Protocols

References

Resources

Orders

Biobricks Used

Gels

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Contents 1 Week 1 1.1 Tuesday 3rd July 2007 1.2 Wednesday 4th July 2007 1.3 Thursday 5th July 2007 1.4 Friday 6th July 2007

Week 1

Tuesday 3rd July 2007

1. Maija and Christine prepared LB broth and LB agar with Protocol 1. Made 4 batches of plates, 2 with kanamycin and 2 with carbenicillin.

Wednesday 4th July 2007

1. All wetlab researched BioBricks.
2. Reporter constructs and mini-Tn5 stocks looked out.
3. Streaked the following:
   • P. putida PAW 340 pJAK14 (Carb. plate)
   • Tn5 lux AB (Carb. plate)
   • Mini-Tn5 lux AB (Carb. plate)
   • P. fluorescens NCIMB 9815 (Carb. plate)
   • P. putida KT2440 (LB)
   • JM109 pBluescript 5k+ (Carb. plate)
   • Mini-Tn5 Tc (Carb. Plate)
   • pQF52 (Carb. plate)
   • P. fluorescens 9815 (LB)
   • E. coli pJAK14 (Km plate)
   • Il DntR in pOF52 (Carb. plate)
   • pUCINR in Ω strain C (Carb. plate)
   • pGLTUR (Carb. plate)
   • Mini-Tn5 Kan (Carb. plate)
   • Mini-Tn5 Sm/Sp (Carb. plate)
   • Mini-Tn5 1cc2 (Carb. plate)
   • E. coli Sa1 (LB)
   • DmpR #24 (Carb. plate)
   • Mini-Tn5 lac 32 in E. coli 517 (Carb. plate)
   • Mini-Tn5 Tc (Carb. Plate)
   • Mini-Tn5 Cm (Carb. plate)
   • DmpR WT (Carb. plate)
   • E.coli sm 10 pESD15 Tn5 GFP (Carb. plate)
   • pUJ8 (Carb. Plate)

Thursday 5th July 2007

1. BioBricks – Maija and Scott transformed using Protocol 2.

   BioBrick Part	Function	Chassis	Plasmid	Kit Plate Location (Plate/Well)
   [http://partsregistry.org/Part:BBa_P1010 Bba_p1010]	Construction plasmid	E. coli DB3.1	pSB1A10	4/7A
   "	"	"	pSB1A10	4/11E
   "	"	"	pSB3K3	4/11C
   [http://partsregistry.org/Part:BBa_I52001 Bba_I52001]	High copy-number construction plasmid	E. coli TOP10 should have been DB3.1	pSB4A5	4/5I
   "	"	"	pSB4K5	4/5D
   "	"	"	pSB4K5	4/6D
   "	"	"	pSB3K5	4/6B
   "	"	"	pSB4A5	4/6E
   [http://partsregistry.org/Part:BBa_J23119 Bba_J23119]	Strong constitutive promoter	E. coli TOP10	pSB1A2	3/19A
   [http://partsregistry.org/Part:BBa_R0062 Bba_R0062]	HSL & LuxR-inducible promoter	E. coli TOP10	pSB1A2	1/9G
   [http://partsregistry.org/Part:BBa_J04500 Bba_J04500]	IPTG-inducible promoter + RBS	E. coli TOP10	pSB1AK3	1/16P
   [http://partsregistry.org/Part:BBa_E0040 Bba_E0040]	Promoter-less GFP	E. coli TOP10	pSB1A2	1/5H

2. Maia and Christine researched restriction enzymes for the reporter constructs and Mini-Tn5s streaked on 4/7/07 and digested some. Had planned to make biobricks using Mini-Tn5s etc but discovered that [http://partsregistry.org/Part:BBa_J61206 a previous attempt to use transposable elements to make biobricks] had been unsuccessful due to scarring at the restriction site.

Friday 6th July 2007

1. Maija and Christine made 10x stocks for M9 (see Protocol 3.2 - M9).
2. Tutorial in [http://www.mbio.ncsu.edu/BioEdit/bioedit.html BioEdit] and [http://biotools.umassmed.edu/bioapps/primer3_www.cgi Primer3].
3. Started designing primers for amplification and site directed mutagenesis of DmpR and DmpR #24 (see Protocol 4).

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