Glasgow/Wetlab/Week5

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Latest revision as of 15:32, 25 October 2007

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Week 5

Monday 30th July 2007

1. Minipreps done (see Protocol 5):

   Gene	Label/Biobrick ID	Primers	Colonies
   	Constructor 4/7A		1, 2, 3, 4, 5, 6, 7, 8, 9
   	Constructor 4/11C		1
   	Constructor 4/11C		10 – 13 did not grow overnight
   	Constructor 2/23N		26, 27, 28, 32
   	Constructor 3/20G		29, 30, 31, 33, 34, 35
   PhzM	B1		1, 2
   PhzS	B3		1, 2, 3
   PhzS		Bbp_PhzS_for_1 and Bbs_Oxy_2	20, 21, 22
   PhzM		Bbp_PhzM_for_1 and Bbs_PhzM_rev_1	23, 24, 25
   PhzS		Bbp_Oxy_1 and Bbs_Oxy_2	14, 15, 16
   PhzM		Bbp_Methyl_1 and Bbs_Methyl_2	17, 18, 19




2. PCR to check if the BioBrick transformants contain the expected size inserts (used VF2 and VR primers) with Reddymix and Touch 2 program (see Protocol 9). Tested:

   Label	4/11C	4/7A	4/5I	4/5O	4/6B	3/19A	1/9G	1/16P	1/5H	B1	B3	B5
   Colonies	10, 11, 12	5, 6, 7	1, 2, 3	1, 2	1, 2, 3	1, 2, 3, 4	1, 2, 3	1, 2, 3	1, 2, 3	17, 18, 19, 25, 26, 27	20, 21, 22, 23, 24	14, 15, 16, 28, 29




3. Diluted new primers which arrived according to the labels.
4. Ran new PCR testing the XylR, Pr and Pu primers (original and new which arrived 30/7/07) on mt-2 with Reddymix and Touch2. Both sets of primers successful. The pGLTUR stock was unreliable.
5. PCR of the 7 gene operon with Reddymix, Touch 2 with an extension time of 8 mins and Pseudomonas Aeruginosa DNA as template. No desired bands seen. Primer combinations as follows:
• Bbp_PhzA_for and Bbs_PhzG_rev
• Bbp_PhzA_for and Bbs_PhzD_rev
• Bbp_PhzD_for and Bbs_PhzG_rev
6. Restriction digest (see Protocol 7) of 3/20G and 2/23N. Used Promega's AvaI for both digests and buffer B, 1hr at 37°C. Ran 10ul of the reaction with 2ul of loading dye on a 1% agarose gel. No bands showed up on the gel picture.

   Label	BioBrick Description	Colonies	Expected Bands
   3/20G	[http://partsregistry.org/Part:BBa_P1010 Bba_p1010] in pSB1AC3 V1005	29, 30, 31, 33, 34, 35	1462bp, 1376bp, 892bp
   2/23N	[http://partsregistry.org/Part:BBa_P1010 Bba_p1010] in pSB1AK3 V1005	26, 27, 28, 32	1462bp, 1376bp, 569bp, 457bp

7. To understand why restriction digests did not appear, ran gel of miniprepped DNA. No bands present. Minipreps ran:
   • 2/23N: Colonies 26, 27, 28
   • 3/20G: Colonies 30, 31
8. Cleaned out cells and reassembled according to NCBE protocol, using one as a negative control replacing yeast with water.

Tuesday 31st July 2007

1. Cells reassembled because they had leaked over night.
2. PCR (see Protocol 9) of the 7 gene operon repeated using KOD
3. polymerase and KOD program. Amplified A-D 3.5kb and D-G 3.5kb. Primer pair as follows:
   • Bbp_PhzA_for and Bbs_PhzG_rev
   • Bbp_PhzA_for and Bbs_PhzD_rev
   • Bbp_PhzD_for and Bbs_PhzG_rev
4. PCR (see Protocol 9) of the 7 gene operon repeated using KOD XL polymerase and KOD XL program. Amplified A-G 7kb. Primer pair as follows:
   • Bbp_PhzA_for and Bbs_PhzG_rev
   • Bbp_PhzA_for and Bbs_PhzD_rev
   • Bbp_PhzD_for and Bbs_PhzG_rev
5. Gel ran of yesterday's BioBrick confirmation (VF2 and VR) PCR. Expected sizes and results as follows:
   

   Label

   4/11C

   4/7A

   4/5I

   4/5O

   4/6B

   3/19A

   1/9G

   1/16P

   1/5H

   B1

   B3

   B5

   Expected Sizes

   981bp

   4096bp

   1396bp

   1396bp

   1396bp

   273bp

   293bp

   535bp

   958bp

   1072bp

   1262bp

   1260bp

   Results

   Correct

   All wrong

   Correct

   Correct

   2 and 3 correct

   Correct

   Correct

   Correct

   Correct

   17, 18, 19, 26 correct

   23 correct

   All wrong

6. Restriction digests of constructors 2/23N and 3/20G carried out again using Roche enzymes (see Protocol 7), Buffer B and incubation for at least 1hr at 37°C. All bands on gel are between 2 and 2.5 kb. Digests as follows:
   

   Constructor	Colonies	Enzymes	Expected Sizes
   2/23N in pSB1AK3	26, 27, 32	BamHI, XhoI	1046bp, 1026bp, 1792bp
   3/20G in pSB1AC3	29, 30, 31, 33, 34, 35	BamHI, XhoI	1046bp, 892bp, 1792bp

7. Overnights set up to test resistances of the 2/23N and 3/20G constructors. All colonies 26-35 grown on carbenicillin LB, and colonies 31, 33 and 34 also grown in chloramphenicol LB.

Wednesday 1st August 2007

1. Meeting with modellers about biobricks.
2. Of yesterday's overnights, the 3 with chloramphenicol did not grow. This confirms that 3/20G is 3/20G and not 2/2B as previously believed as a result of mislabelling. All overnights then miniprepped according to Qiagen protocol.
3. New restriction digest for new miniprepped 3/20G and 2/23N. 3/20G using Roche enzymes, Buffer B and incubating for at least 1hr at 37°C, digestion worked. 2/23N using Promega's AvaI restriction enzyme as on 30/7/07, AvaI cut the plasmid but not the biobrick.
   • 3/20G in pSB1AC3, colonies 30 and 31: BamHI, PvuI – 1411bp, 1343bp (Xho does not cut).
   • Overnights set up: 2/23N – 26, 27, 28 and 3/20G – 33 (all carb).
4. Gel run of 7 gene operon PCR from 31/7/07 and DNA extracted according to Qiagen Gel Extraction Microcentrifuge Protocol (see Protocol 11).
5. PCR (see Protocol 9) repeated for XylR, Pr and Pu because the previous Pu primer combinations did not work. Primer combinations as follows:
   • XylR preffix and XylR suffix
   • Pr prefix and Pr suffix
   • Pr_for_2 and XylR_rev_2
   • Pu_Pre_EM and Pu_Suf_AFT
   • Pu_Pre_AFT and Pu_Suf_EM
6. Researched restriction maps for plasmids with death genes.
7. Overnights set up with the intention of digesting constructors of PhzM and PhzS after miniprepping in the morning, hopefully getting ligations that will transform TOP10 cells to grow overnight. Colonies were chosen that gave a positive result of the right predicted size (see 31/7/07) on a gel of PCR with VF2 and VR primers.
   • 4/11C constructor – 10, 11, 12 (Kan)
   • 4/5I constructor – 1, 2, 3 (Carb)
   • 4/5O constructor – 1, 2, 3 (Kan)
   • 4/6B constructor – 2, 3 (Kan)
   • B1 PhzM - 17, 18, 19, 26 (Carb)
   • B3 PhzS - 20 (Carb)
   • C5 PhzM - 24, 25 (Carb)
8. Miller Assay was performed (according to Protocol 10) on E. coli cell culture aliquots containing the DntR system grown up in LB. A range of salicylate concentrations (0 mM, 20 uM, 50 uM, 100 uM and 200 uM)were added to aliqouts and then allowed to grow for a further 3 hours. As this was a trial run, measurements at only one time point (58 minutes) were taken. The spectrophotometer readings and their corresponding calculated Miller units are shown in the table below.

The very high level of LacZ expression throughout the range of concentrations of salicylate could be due to the content of tryptophan in the LB media which could act upon the DntR responsive promoter. In order to try and minimize the background LacZ expression due to the presence of aromatic compounds present in the LB media, cell cultures will be grown up in minimal media instead.

Thursday 2nd August 2007

1. Minimal media prepared for the Miller Assay because it does not interfere with enzyme activity (see Protocol 10).
2. Minipreps (see Protocol 5) done of yesterday's overnights for PhzM and PhzS.
3. Digestions (see Protocol 7) done of these PhzM and PhzS minipreps:

4. PCR (see Protocol 9) done with newly arrived death gene primers on the miniprepped DNA to make sure the BioBrick overnights genuinely carry their respective versions of ccdB (esp 23N as it does not run correctly on the gel) using Reddymix and Touch 2 with Tm of 60C and 28 cycles, saved as PCR60DEG.

5. PCR (see Protocol 9) also done (using the primers used to create the cloned versions of PhzM and PhzS) on minipreps, done this morning, of TOPO cloning (hopefully containing PhzM and PhzS) to confirm that the insert is genuinely there. Used Reddymix and Touch2. All constructs appear to contain the correct genes.

Friday 3rd August 2007

1. Miller assay (see Protocol 10) repeated on E.coli cultures grown up in minimal media. Measurements were taken at three time points (10, 30 and 60 minutes) and the range of salicylate concentrations used was expanded (0mM, 10 uM, 100 uM, 1mM and 10 mM)

1. A 1% gel was run for the p1010 PCR products from 02/08.
   • 23N (pSB1AK3) plasmid size ~3800bp - AvaI did not cut the biobrick.
   • 20G (pSB1A2) plasmid size ~2700bp - digest worked!

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