Glasgow/Wetlab/Week4

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Latest revision as of 17:08, 22 October 2007

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Week 4

Monday 23rd July 2007

1. PCR to amplify PhzM and PhzS Program "Touch 2" with [[User:EmmaTravis|Emma]'s primers (see Orders 1) using Reddy Mix (see Protocol 9) and Pseudomonas Aeruginosa 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. Phz_S_for_1 and Phz_S_rev_1
   6. Bbp_Phz_S_1 and Bbp_Phz_S_1
   7. Phz_M_for_1 and Phz_M_rev_1
   8. Bbp_Phz_M_for_1 and Bbp_Phz_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 Aeruginosa as our template DNA. The primer pairs used:
   1. PhzUp and PhzLow
   2. bbpPhzUp and bbsPhzLow
   3. bbpPhzUp and PhzE_SDM_EcoRI_rev
   4. bbsPhzLow and PhzB_SDM_EcoRI_for
3. PCR with functioning DntR primers using KOD polymerase and KOD Program (see 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 24th July 2007

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:
   • bbpPhzUp and PhzB_SDM_EcoRI_rev
   • PhzB_SDM_EcoRI_for and PhzE_SDM_EcoRI_rev

And for the KOD polymerase reaction also:

1. • bbsPhz_Low and PhzB_SDM_EcoRI_for
2. Repeated PCR reaction to amplify PhzM and PhzS using KOD polymerase and KOD program (see Protocol 9). Results show that bbsPhzS_rev_1 is faulty, will redesign. Primer combinations also included:
   • PhzS_for_1 and bbsPhzS_rev_1
   • bbp_PhzS_for_1 and PhzS_rev_1
3. Gel-extracted from the PCR to amplify PhzM and PhzS, 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 25th July 2007

1. Overnights of pQF52 did not grow, so done again.
2. Overnights also set up for the [http://partsregistry.org/Part:BBa_P1010 BBa_p1010] death gene biobrick in pSB3K3to see if cloning could be made faster by inserting our parts into construction plasmids directly from PCR products.
3. Transformations for PhzM and PhzS 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 PhzM and PhzS 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:

   Label	Gene	Primers
   A2	PhzM	Methyl_1 and Methyl_2
   A7	PhzS	bbp_Oxy_1 and bbp_Oxy_2
   B7	PhzM	bbp_PhzM_for_1 and bbs_PhzM_rev_1
   C4	PhzS	bbp_PhzS_for_1 and PhzS_rev_1

5. Restriction digests of [http://partsregistry.org/Part:BBa_P1010 BBa_p1010] in pSB3K3 were redone using Roche recipe (Protocol 7) as previous attempts were unsuccessful.

   Label	Description	Enzymes	Expected Sizes
   4/11C	Death gene plasmid	AraI	274bp, 604bp, 569bp, 1978bp
   4/11C	Death gene plasmid	EcoRI, SpeI	698bp, 2727bp
   4/11C	Death gene plasmid	BamHI, XhoI	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 PhzD, and forward and reverse primers for PhzA and PhzG respectively.

Thursday 26th July 2007

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 (see Protocol 10) 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 PhzD, and forward and reverse primers for PhzA and PhzG 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. PhzD site directed mutagenesis primers, and PhzA forward and PhzG 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 pCR4 vector).
10. PCR with KOD on various combinations of the PhzM and PhzS 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_PhzS_rev_1 fail. Primer combinations as follows:

    Label	Primers	PCR Program
    A1	Bbp_PhzM_for_1 and bbs_PhzM_rev_1	Gradient program 58-65ºC
    A3	Bbp_PhzS_for_1 and BbsPhzS_rev_1	Gradient program 58-65ºC
    A5	Bbp_PhzS_for_1 and PhzS_rev_1	Gradient program 58-65ºC
    A7	Bbp_Methyl_1 and Bbs_Methyl_2	Touch 2
    B1	Bbp_PhzM_for_1 and Bbs_Methyl_2	KOD
    B3	Bbp_PhzS_for_1 and Bbs_Oxy_2	KOD
    B5	Bbs_Oxy_1 and Bbs_Oxy_2	KOD
    B7	Bbp_Oxy_1 and BbsPhzS_rev_1	KOD
    C1	Bbp_Methyl_1 and bbs_PhzM_rev_1	KOD
    C3	Bbp_PhzS_for_1 and BbsPhzS_rev_1	KOD
    C5	Bbp_PhzM_for_1 and bbs_PhzM_rev_1	KOD
    C7	Bbp_Methyl_1 and Bbs_Methyl_2	KOD

11. B1, B3, B5, C5 and A7 were gel-extracted or PCR-purified then ligated into TOPO pCR4 vector before transforming into TOP10 cells.
    • 'Loose' M, A7: Bbp_Methyl_1 and Bbs_Methyl_2
    • 'Close' M, B1: Bbp_PhzM_for_1 and Bbs_Methyl_2
    • 'Close' S, B3: Bbp_PhzS_for_1 and Bbs_Oxy_2
    • 'Loose' S, B5: Bbs_Oxy_1 and Bbs_Oxy_2
    • 'Tight' M, C5: Bbp_PhzM_for_1 and bbs_PhzM_rev_1

Friday 27th July 2007

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 pCR4 vector successful, DntR DNA to be sent for sequencing.

   Enzyme	PvuI	BamHI, NcoI
   Expected Fragments	878bp, 2175bp, 1851bp	2280bp, 2624 bp
   Observed Fragments	878bp, 2175bp, 1851bp	2280bp, 2624bp

   
   

5. DntR overnights for the Miller Assays (see Protocol 10) finally grew. To be subcultured on Sunday for use on Monday.
6. Construction vectors results (26/07/07 (11.))
   • 4/11E [http://partsregistry.org/Part:BBa_P1010 BBa_p1010] in pSB1A10 - no growth
   • 4/11C [http://partsregistry.org/Part:BBa_P1010 BBa_p1010] in pSB3K3 - 1 colony - inoculated overnight
   • 4/7A [http://partsregistry.org/Part:BBa_P1010 BBa_p1010] in pSB1A10 - 2 colonies - inoculated overnight
   • B1: Bbp_PhzM_for_1 and Bbs_Methyl_2 - 2 colonies - inoculated overnight
   • B3: Bbp_PhzS_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_PhzM_for_1 and bbs_PhzM_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 PhzM and PhzS 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 [http://partsregistry.org/Part:BBa_P1010 BBa_p1010] in pSB1AK3 V1005
   • 2/23P [http://partsregistry.org/Part:BBa_P1010 BBa_p1010] in pSB1AT3 V1005
   • 2/2B [http://partsregistry.org/Part:BBa_P1010 BBa_p1010] in pSB1AC3 V1005
   • 3/20G [http://partsregistry.org/Part:BBa_P1010 BBa_p1010] in pSB1A2 V1015
   
   

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