Glasgow/Wetlab

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PROTOCOLS REFERENCES ORDERS

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:
    • 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)

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).
  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).

Week 2

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 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 and restriction digested 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 Top 10s, not so much DB3.1). To be mini-prepped tomorrow.

11/07

  1. Lynsey mini-prepped Scott's retransformed biobricks (9/7/07) according to Qiagen Prepkit Manual.
  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.

12/07

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 BlastX 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.

13/07

  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, Majeik, Toby, Christine M, Mai, Scott and Lynsey.

Week 3

17/07

  1. Restriction Digests:
    • BBa_J23119 (strong constitutive promoter) pSB1A2. 1 x NheI, 1 x PvuI. 680bp, 1430bp
    • BBa_R0062 (HSL and luxR) pBB1A2. 1 x EcoRI, 1 x PvuI. 1460bp, 660bp
    • BBa_J04500 (IPTG inducer and RBS) pSB1AK3. 1 x PvuII, 2 x PvuI. 2250bp, 1030bp, 730bp
    • BBa_p1010 (death gene) pSB3K3. 1 x BamHI, 2 x XhoI. 190bp, 2390bp,840bp.
    • BBa_E0040 (GFP no promoter) pSB1A2. 1 x Hime II, 1 x PvuI. 1630bp, 1170bp.
    • BBa_I52001 (6D) p5B4K5. 2 x AvaI, 1 x PvuI. 920bp, 1470bp, 2120bp.
    • See Protocol 7 for Restriction Digests.

18/07

  1. Last night we diluted primers to 100 pmol/ul by adding the amount of dH2O given on the order sheet, and made working solutions to 10 pmol/ul (2 x 200 ul each - 20 ul stock and 180 ul dH2O).
  2. Maija ran a gel of the restriction digests to check the sizes of the biobricks. These were the maps we made yesterday.
  3. Mai is extracting DNA from 3 samples of ****, which were plated yesterday and grown in liquid media, using MoBio PowersoilTM DNA purification kit (see Prorocol 8), only change was to shorten "shaking" time from 10 to 2 minutes.
  4. PCR trial run using Reddymix and Touch 2 done on:
    • pGLTUR: XylR_prefix / XylR_suffix, Pr_prefix / Pr_suffix, Pr_prefix / XylR_suffix, Pu_prefix_EM / Pu_suffix_EM.
    • pQF52 plasmid: DntR_prefix / DntR_suffix
    • DmpR WT/24: DntR_prefix / DntR_suffix
    • Biobricks: R0062, E0040, J04500, J23119, p1010, I52001.
    • See Prorocol 9 for PCR.

19/07

  1. Mai redid PCR on pGLTUR and pQF52 using Reddymix and a gradient program (See Prorocol 9.1) to minimise the non-specific amplification evident in previous gels. New DntR suffix primer has arrived. Used the following primer sets.
    • XylR prefix and XylR suffix
    • Pr prefix and Pr suffix
    • Pr prefix and XylR suffix
    • Pu prefix and Pu suffix
    • DntR prefix and DntR suffix 2

20/07

Week 4

23/07

  1. PCR Program "Touch 2" with Emma's primers (see Orders 1) using Reddy Mix (See Protocol 9.1) and Pseudomonas as our template DNA. The primer pairs used:
    1. Methyl_1 and Methyl_2
    2. Oxy_1 and Oxy_2
    3. Bbp_Methyl_1 and Bbp_Methyl_2
    4. Bbp_Oxy_1 and Bbp_Oxy_2
    5. (***)_S_for_1 and (***)_S_rev_1
    6. Bbp_(***)_S_1 and Bbp_(***)_S_1
    7. (***)_M_for_1 and (***)_M_rev_1
    8. Bbp_(***)_M_for_1 and Bbp_(***)_M_rev_1
  2. PCR of 7 gene operon using Program "Touch 2" with extension time of 8 mins with Emma's primers (see Orders 1) and site directed mutagenesis primers (see Orders 2) using Reddy Mix (See Protocol 9.1) and Pseudomonas as our template DNA. The primer pairs used:
    1. (***)Up and (***)Low
    2. bbp(***)Up and bbs(***)Low
    3. bbp(***)Up and (***)E_SDM_EcoRI_rev
    4. bbs(***)Low and (***)B_SDM_EcoRI_for
  3. KOD polymerase PCR on functioning DntR primers using KOD polymerase Protocol 9)and KOD Program Protocol 9).
  4. Redid PCR for XylR, Pr and Pu as from 19/07/07 from glycerol stocks, DNA from plates and colony PCR.
  5. Set up overnights of pGLTUR and pQF52 to do lux and lacZ assays.

24/07

  1. Redid 7 gene operon PCR (23/07/07 (2)) with Reddymix and Touch 2, and then redid again with KOD polymerase and KOD program with extension time of 2 mins, 55ºC annealing temperature, and 40 cycles Protocol 9). Added the extra primers combinations for both reactions:
    • bbp(***)Up and (***)B_SDM_EcoRI_rev
    • (***)B_SDM_EcoRI_for and (***)E_SDM_EcoRI_rev
      • And for the KOD polymerase reaction also:
    • bbs(***)_Low and (***)B_SDM_EcoRI_for
  2. Repeated PCR reaction to amplify (*m*) and (*s*) using KOD polymerase and KOD program (see Protocol 9). Results show that bbs(*s*)_rev_1 is faulty, will redesign. Primer combinations also included:
    • (*s*)_for_1 and bbs(*s*)_rev_1
    • bbp_(*s*)_for_1 and (*s*)_rev_1
  3. Gel-extracted from the PCR to amplify (*M*) and (*s*), and purified in order to transform ( Protocol 2) into Top10 cells. Transformations did not work, will do again tomorrow.
  4. Colony PCR for XylR, Pr and Pu (23/07/07 (4.)) showed no unique bands in any of the primer pairs. Will redesign primers for pGLTUR.

25/07

  1. Overnights of pQF52 did not grow, so done again.
  2. Overnights also set up for the Bba_p1010 death gene biobrick to see if cloning could be made faster by inserting our biobrick constructs into the biobrick plasmid.
  3. Transformations for (*m*) and (*S*) done again (24/07/07 (3.)) and overnights set up.
  4. The rest of the KOD PCR (24/07/07 (2.)) products were run on gel in order to PCR amplify the (*M*) and (*S*) genes (not gel extraction because A overhangs are required for 7A cloning). From the results, another PCR is required for B7 and C4 over a gradient. Primer pairs used:
    • A2: Methyl_1 and Methyl_2
    • A7: bbp_Oxy_1 and bbp_Oxy_2
    • B7: bbp_(*m*)_for_1 and bbs_(*m*)_rev_1
    • C4: bbp_(*s*)_for_1 and (*s*)_rev_1
  5. Restriction digests of Bba_p1010 pSB3K3 were redone using Roche recipe (Protocol 7) as previous attempts were unsuccessful.
    1. AraI, predicted fragment sizes 274bp, 604bp, 569bp, 1978bp.
    2. EcoRI and SpeI, predicted fragment sizes 698bp, 2727bp.
    3. BamHI, XhoI, predicted fragment sizes 188bp, 843bp, 2394bp.
  6. An alternative source of the XylR gene, P. Putida mt-2 strain, which contains the TOL plasmid plated out and overnights made up (28ºC).
  7. PCR of 7 gene operon does not show bands of interest, will redesign primers (see Orders 2) for site directed mutagnesis on (*d*), and forward and reverse primers for (*a*) and (*g*) respectively.
  8. PCR with KOD on various combinations of the (*m*) and (*s*) gene primers with different PCR programs (Protocol 7) with the intention of cloning them into TOPO TA vectors and BB construction vectors. A1 and A7 gave good bands but thair KOD equivalents C1 and C7 gave a closer match. Again and combinations with Bbs_(*s*)_rev_1 fail. Primer combinations as follows:
    • A1 Bbp_(*m*)_for_1 and bbs_(*m*)_rev_1, Gradient program 58-65ºC
    • A3 Bbp_(*s*)_for_1 and Bbs(*s*)_rev_1, Gradient program 58-65ºC
    • A5 Bbp_(*s*)_for_1 and (*s*)_rev_1, Gradient program 58-65ºC
    • A7 Bbp_Methyl_1 and Bbs_Methyl_2, Touch 2
    • B1 Bbp_(*m*)_for_1 and Bbs_Methyl_2, KOD
    • B3 Bbp_(*s*)_for_1 and Bbs_Oxy_2, KOD
    • B5 Bbs_Oxy_1 and Bbs_Oxy_2, KOD
    • B7 Bbp_Oxy_1 and Bbs(*s*)_rev_1, KOD
    • C1 Bbp_Methyl_1 and bbs_(*m*)_rev_1, KOD
    • C3 Bbp_(*s*)_for_1 and Bbs(*s*)_rev_1, KOD
    • C5 Bbp_(*m*)_for_1 and bbs_(*m*)_rev_1, KOD
    • C7 Bbp_Methyl_1 and Bbs_Methyl_2, KOD

Overall a great success! We have now amplified

  • 'Loose' M, A7: Bbp_Methyl_1 and Bbs_Methyl_2
  • 'Close' M, B1: Bbp_(*m*)_for_1 and Bbs_Methyl_2
  • 'Close' S, B3: Bbp_(*s*)_for_1 and Bbs_Oxy_2
  • 'Loose' S, B5: Bbs_Oxy_1 and Bbs_Oxy_2
  • 'Tight' M, C5: Bbp_(*m*)_for_1 and bbs_(*m*)_rev_1

26/07

  1. Minipreps of DntR transformant and death gene plasmid overnights done (see Protocol 5).
  2. Digests of death gene plasmid show the wrong sizes of bands (see 25/07/07 (2.)).
  3. Overnights of pQF52 with the DntR gene for the Miller assay have still not grown, new overnights set up.
  4. mt-2s have not grown overnight either, possibly because they were done from glycerol stocks. New colonies set up with colonies from LB plates.
  5. PCR of 7 gene operon does not show bands of interest, will redesign primers (see Orders 2) for site directed mutagnesis on (*d*), and forward and reverse primers for (*a*) and (*g*) respectively.
  6. Redesigned XylR, Pr and Pu primers using sequence for XylR (Iouye et al, 1988) and located Pr and Pu sequences using predesigned primer sequences (Willardson et al, 1998) (Orders 2).
  7. New primers also designd for the death gene plasmid (Orders 2).
  8. (*d*) site directed mutagenesis primers, and (*a*) forward and (*g*) reverse primers designed.
  9. PCR done on DntR transformants (2 colonies) using Reddymix and Touch 2 with extension time of 1min 30secs (see Protocol 9). PCR transformants give the right sizes of bands (M13 rev starts a little outside the mcs of the TOPO vector).

27/07

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