Glasgow/Wetlab

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PROTOCOLSREFERENCES

Contents

Week 1

03/07

  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.

04/07

  1. All wetlab researched BioBricks.
  2. Reporter constructs and mini-Tn5 stocks looked out.
  3. Streaked the following:
    1. P. putida PAW 340 pJAK14 (Carb. plate)
    2. Tn5 lux AB (Carb. plate)
    3. Mini-Tn5 lux AB (Carb. plate)
    4. P. Fluorescens NCIMB 9815 (Carb. plate)
    5. P. putida KT2440 (LB)
    6. JM109 pBluescript 5k+ (Carb. plate)
    7. Mini-Tn5 Tc (Carb. Plate)
    8. pQF52 (Carb. plate)
    9. P. Fluorescens 9815 (LB)
    10. E. coli pJAK14 (Km plate)
    11. Il DntR in pOF52 (Carb. plate)
    12. pUCINR in Ω strain C (Carb. plate)
    13. pGLTUR (Carb. plate)
    14. Mini-Tn5 Kan (Carb. plate)
    15. Mini-Tn5 Sm/Sp (Carb. plate)
    16. Mini-Tn5 1cc2 (Carb. plate)
    17. E. coli Sa1 (LB)
    18. DmpR #24 (Carb. plate)
    19. Mini-Tn5 lac 32 in E. coli 517 (Carb. plate)
    20. Mini-Tn5 Tc (Carb. Plate)
    21. Mini-Tn5 Cm (Carb. plate)
    22. DmpR WT (Carb. plate)
    23. E.coli sm 10 pESD15 Tn5 GFP (Carb. plate)
    24. pUJ8 (Carb. Plate)

05/07

  1. BioBricks – Maija and Scott transformed using Protocol 2.
    • BBa_p1010 (DB3.1) (death gene plasmid) Plate 4: 7A – p5B1A10, 11E – p5B1A10 and 11C – p5B3K3
    • BBa_IS2001 (Top10) (high copy number plasmid) Plate 4: 5I – p5B4A5, 5D – p5B4K5, 6B – p5B3K5, 6D – p5B4K5 and 6E – p5B4A5 (Also 5K, 5M, 4J, 4L, 4N, 4P)
    • BBa_J23119 (Top 10) (strong constitutive promoter) Plate 3: 19A – pB1A2 (V1013)
    • BBa_R0062 (Top 10) (HSL and luxR inducible) Plate 1: 9G – pSB1A2 (V1004)
    • BBa_J04500 (Top 10) (IPTG inducible prom + RBS) Plate 1: 16P – p5B1AK3 (V1009)
    • BBa_E0040 (Top 10) (GFP mutant no promoter (3b)) Plate 1: 5H – p5B1A2 (V1001)
  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 a previous attempt to use transposable elements to make biobricks had been unsuccessful due to scarring at the restriction site. (http://partsregistry.org/Part:BBa_J61206)

06/07

  1. Christine and Maija designed primers for site directed mutagenesis in DmpR, DmpR #24, PhzM, PhzS and PhzABCDEFG, and amplification of DmpR and DmpR #24. Used Protocol 4, and to check Tm http://www.itt-biotech.de/itt-cgi/oligo-tm.pl
  2. Maia carried out restriction digests of DmpR and DmpR #24. Results were poor and gel gave poor visibility.
  3. Scott retransformed any of the transformations that did not work from the transformations from 5/7/07. #*4/11C BBa_p1010 pSB3K3 death gene
    • 4/5I BBa_I522001 pSB4A5 hi-copy

5 4/5D BBa_I522001 pSB4K5 hi-copy 6 4/6B BBa_I522001 pSB3K3 hi-copy 7 4/6D BBa_I522001 pSB4K hi-copy 8 1/5H BBa_E0040 pSB1A2 GFP non promoter 9 Used transformations that did work and set them up tubes of LB for minipreps tomorrow. 10 BBa_I52001 death gene plasmid (hi copy number) 11 BBa-J23119 strong constitutive promoter 12 BBa_R0062 HSL and luxR inducible 13 BBa_306500 IPTG inducible and RBS Plan is to use DmpR and DmpR #24 to detect phenol and produce lacZ. Grown on Xgal the better the bacteria detect phenol, the more blue they will be in a spectrophotometer.

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