Glasgow/Wetlab/Week2

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Revision as of 12:56, 28 September 2007

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Contents

Week 2

Monday 9th July 2007

  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/iit-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 X-gal, the better the bacteria detect phenol, the more blue they will be in a spectrophotometer.

Tuesday 10th July 2007

  1. Maia did minipreps, according to Qiagen 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 50 ml 100% ethanol to make 5 mg/ml stock.
  4. Maija made thiamine stock (0.8 g thiamine in 20 ml 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 (see Protocol 5) and restriction digested (see Protocol 7) tomorrow.
    • 2x 5ml LB containing carb (50 µg/ml) with DmpR
    • 2x 5ml LB containing carb (50 µg/ml) with DmpR #24
    • 2x 5ml LB containing Tc (50 µg/ml) with DmpR
    • 2x 5ml LB containing Tc (50 µg/ml) with DmpR #24
    • 2x 5ml LB containing Tc (10 µg/ml) with DmpR
    • 2x 5ml LB containing Tc (10 µg/ml) with DmpR #24
  7. Scott's retransformations (9/7/07) all worked (esp TOP10s, not so much DB3.1). To be mini-prepped tomorrow (see Protocol 5).

Wednesday 11th July 2007

  1. Lynsey mini-prepped Scott's retransformed biobricks (9/7/07) according to Qiagen Prepkit Manual (see Protocol 5).
  2. Maija prepared glycerol for freezing the transformations in LB.
  3. Can not mini-prep DmpR or DmpR #24 in Tc because it did not grow well overnight. Time for Plan B.
    • Plan B
      DmpR is not working – not growing or digesting as we would expect it to. Instead of DmpR we will try XylR which detects BETX compounds (benzene, toluene, and xylene) and DntR which detects salicylate and could be modified to detect TNT and DNT. For this we will be using pGLTUR and pQF52.
  4. We all began to design primers for site directed mutagenesis (SDM) and amplification of XylR, Pr, Pu and DntR.

Thursday 12th July 2007

XylR, Pr, and Pu

  1. Searched [http://www.ncbi.nlm.nih.gov/Genbank GenBank] for pWW0 which contains XylR, Pr and Pu. Saved in BioEdit.
  2. Using the XylR sequence (Inouye et al, 1988) we were able to design primers for the amplification of XylR.
  3. Using previously designed primers (Willardson et al, 1998) we were able to locate the beginning of Pr and designed primers to amplify the sequence between the beginning of Pr and the beginning of XylR.
  4. From previously designed primers (Willardson et al, 1998) we were able to locate a sequence we believed to be Pu and designed primers. To be sure we also searched pWW0 sequence with [http://biotools.umassmed.edu/bioapps/primer3_www.cgi Primer3] to locate on the plasmid where the Willardson Pu primers would attach. Using BioEdit we located another sequence we also suspect to be Pu. We now have primers designed for both suspected sequences.
  5. Also designed primers for site directed mutagenesis of the PstI site in XylR.

(We were unable to use the Willardson primers for our purposes because they were designed to contain restriction sites, instead we used them to locate the genes of interest).

DntR

  1. Scott found an article containing the sequence for DntA and some of DntR, then we used [http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?PAGE=Nucleotides&PROGRAM=blastn&MEGABLAST=on&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on Blast] to find the sequence of DntR. From this we were able to design primers to amplify the sequence.
  2. Also designed primers so we can sequence pQF52 because the lacZ gene is not complete in the plasmid and we need to know its sequence.

Friday 13th July 2007

  1. Began typing up protocols for the Wiki.
  2. Ordered primers, made changes to DntR_suffix_1 which will arrive later. (See Orders)
  3. Wiki meeting. Maija, Christine H, Maciej, Toby, Christine M, Mai and Lynsey.


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