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. Maija and Christine made 10x stocks for M9 (see Protocol 3).
  2. Tutorial in BioEdit and Primer3.
  3. Started designing primers for amplification and site directed mutagenesis of DmpR and DmpR #24 (see Protocol 4).

09/07

  1. Christine and Maija designed primers for site directed mutagenesis in DmpR, DmpR #24, ****, **** and ****, 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. Mai 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
    • 4/5D BBa_I522001 pSB4K5 hi-copy
    • 4/6B BBa_I522001 pSB3K3 hi-copy
    • 4/6D BBa_I522001 pSB4K hi-copy
    • 1/5H BBa_E0040 pSB1A2 GFP non promoter
  4. Used transformations that did work and set them up tubes of LB for minipreps tomorrow.
    • BBa_I52001 death gene plasmid (hi copy number)
    • BBa-J23119 strong constitutive promoter
    • BBa_R0062 HSL and luxR inducible
    • 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.

10/07

  1. Mai did minipreps, according to Quiagen prepkit manual (see Protocol 5), of the transformations grown in LB last night (9/7/07).
  2. Wetlab and Drylab gave presentations to the team to explain key terms used in the lab (see Tutorials).
  3. Christine made tetracycline stock – 250 mg tetracycline in 50ml 100% ethanol to make 5mg/ml stock.
  4. Maija made thiamine stock (0.8g thiamine in 20ml dH2O and filter sterilized. Kept in freezer in foil (light sensitive).
  5. Christine grew E. coli pJAK14 in LB overnight at 37°C following protocol from Wise et al, 2000).
  6. Scott and Lynsey grew DmpR and DmpR #24 overnight in LB with Tc and Carb. This is because the plates streaked on 4/7/07 for DmpR and DmpR #24 did not grow, and restriction digests on 9/7/07 used up most of the DNA. What is grown will be mini-prepped and restriction digested tomorrow.
    • 2x 5ml LB containing carb (50ug/ml) with DmpR
    • 2x 5ml LB containing carb (50ug/ml) with DmpR #24
    • 2x 5ml LB containing Tc (50ug/ml) with DmpR
    • 2x 5ml LB containing Tc (50ug/ml) with DmpR #24
    • 2x 5ml LB containing Tc (10ug/ml) with DmpR
    • 2x 5ml LB containing Tc (10ug/ml) with DmpR #24
  7. Scott's retransformations (9/7/07) all worked (esp Top 10s, not so much DB3.1). To be mini-prepped tomorrow.
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