Glasgow/Wetlab/Week4

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Contents

Week 4

Monday 23/07

  1. PCR Program "Touch 2" with Emma's primers (see Orders 1) using Reddy Mix (See Protocol 9) 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) 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. PCR with functioning DntR primers using KOD polymerase 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.

Tuesday 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.

Wednesday 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 parts into construction plasmids directly from PCR products.
  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 fresh A overhangs are required for TA 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 was 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.

Thursday 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).
  10. PCR with KOD on various combinations of the (*m*) and (*s*) gene primers with different PCR programs (Protocol 9) 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
  11. B1, B3, B5, C5 and A7 were gel-etracted and PCR-purified into TOPO vectors in Top10 cells.
    • '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

Friday 27/07

  1. Order placed for primers (see 26/07/07 (5., 6., and 7.)).
  2. Miniprep done of the mt-2 overnights (1 and 2) according to Qiagen prepkit manual (see Protocol 5).
  3. Further PCR done from mt-2 with the existing primers for the XylR system using Reddymix and Touch 2 (see Protocol 9). Used the following primer pairs:
    • XylR prefix and XylR suffix
    • Pr prefix and Pr suffix
    • Pr prefix and XylR suffix
    • Pu prefix EMMA and Pu suffix EMMA
    • Pu prefix AFTER XylR and Pu suffix AFTER XylR
  4. Restriction Digest of DntR in TOPO vector successful, DntR DNA to be sent for sequencing.
    • PvuI - expected fragments 878bp, 2175bp, 1851bp, - observed fragments 878bp, 2175bp, 1851bp.
    • BamHI and NcoI - expected fragments - 2280bp, 2624 bp, - observed fragments 2280bp, 2624bp.
  5. DntR overnights for the Miller assays finally grew. To be subcultured on Sunday for use on Monday.
  6. Construction vectors results (26/07/07 (11.))
    • 4/11E Bba_p1010 pSB1A10 - no growth
    • 4/11C Bba_p1010 pSB3K3 - 1 colony - inoculated overnight
    • 4/7A Bba_p1010 pSB1A10 - 2 colonies - inoculated overnight
    • B1: Bbp_(*m*)_for_1 and Bbs_Methyl_2 - 2 colonies - inoculated overnight
    • B3: Bbp_(*s*)_for_1 and Bbs_Oxy_2 - 3 colonies - inoculated overnight
    • B5: Bbs_Oxy_1 and Bbs_Oxy_2 - no growth, will try cloning direct from gel extraction into biobrick vector
    • C5: Bbp_(*m*)_for_1 and bbs_(*m*)_rev_1 - no growth, will try cloning direct from gel extraction into biobrick vector
  7. Observation: TOPO pCR4 vector uses disruption of the ccdB gene as a selection method and the PCR transformants were plated on carbenicillin, perhaps this has killed the transformants. Colony PCR will be done on the successful ones. Overnights were then grown from 3 or more colonies from each (*m*) and (*s*) transformation plate on carbenicillin again. Also leftovers from the gel extractions and purifications(26/07/07) were plated on LB only for later analysis.
  8. More constuction vectors (constructors) were selected for their multiple resistances from the registry to transform into DB3.1. These were then diluted from the kit plates and transformed into DB3.1 (using Protocol 2) and plated overnight. They are as follows:
    • 2/23N Bba_p1010 pSB1AK3 V1005
    • 2/23P Bba_p1010 pSB1AT3 V1005
    • 2/2B Bba_p1010 pSB1AC3 V1005
    • 3/20G Bba_p1010 pSB1A2 V1015
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