Glasgow/Wetlab/Week5

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Revision as of 13:01, 28 September 2007

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Protocols References Resources Orders Biobricks
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Contents

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
    (*m*) B1 1, 2
    (*s*) B3 1, 2, 3
    (*s*) Bbp_(*s*)_for_1 and Bbs_Oxy_2 20, 21, 22
    (*m*) Bbp_(*m*)_for_1 and Bbs_(*m*)_rev_1 23, 24, 25
    (*s*) Bbp_Oxy_1 and Bbs_Oxy_2 14, 15, 16
    (*m*) 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 ***** DNA as template. No desired bands seen. Primer combinations as follows:
    • Bbp_(*a*)_for and Bbs_(*g*)_rev
    • Bbp_(a*)_for and Bbs_(*d*)_rev
    • Bbp_(*d*)_for and Bbs_(*g*)_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_(*a*)_for and Bbs_(*g*)_rev
    • Bbp_(a*)_for and Bbs_(*d*)_rev
    • Bbp_(*d*)_for and Bbs_(*g*)_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_(*a*)_for and Bbs_(*g*)_rev
    • Bbp_(a*)_for and Bbs_(*d*)_rev
    • Bbp_(*d*)_for and Bbs_(*g*)_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 (*m*) and (*s*) 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 (*m*) - 17, 18, 19, 26 (Carb)
    • B3 (*s*) - 20 (Carb)
    • C5 (*m*) - 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.



[Salicylate] (uM) Abs600 Abs420 Miller units
Rep A Rep B Rep C Rep A Rep B Rep C Rep A Rep B Rep C
0 0.386 0.480 0.495 0.209 0.257 0.317 466.8 461.6 552.1
20 0.676 0.814 0.473 0.431 0.450 0.364 549.6 476.6 663.4
50 0.494 0.495 0.476 0.380 0.396 0.489 663.1 689.7 885.6
100 0.533 0.464 0.507 0.373 0.357 0.463 603.3 663.3 787.3
200 0.551 0.529 0.521 0.526 0.548 0.451 823.0 893.0 746.2



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 (*m*) and (*s*).
  3. Digestions (see Protocol 7) done of these (*m*) and (*s*) minipreps:


Gene Label Colonies Primers Enzyme Orientation 1 Orientation 2 Results
(*m*) B1 17, 18, 19, 26 (*m*)_for_1 + Methyl_2 PstI 4243bp, 443bp, 275bp 3683bp, 835bp, 443bp 17, 18 + 26 possibly in orientation 1
(*m*) B1 17, 18, 19, 26 (*m*)_for_1 + Methyl_2 EcoRI 3654bp, 1287bp, 18bp 4654bp, 286bp, 18bp 17, 18 + 26 possibly in orientation 1
(*m*) C5 24, 25 (*m*)_for_1 + (*m*)_rev_1 PstI 4243bp, 443bp, 275bp 3683bp, 835bp, 443bp Appears wrong
(*m*) C5 24, 25 (*m*)_for_1 + (*m*)_rev_1 EcoRI 4243bp, 443bp, 275bp 3683bp, 835bp, 443bp Possibly in orientation 1
(*s*) B3 20 (*s*)_for_1 + Oxy_2 PstI 4012bp, 534bp, 344bp, 275bp 3683bp, 604bp, 534bp, 344bp Possibly in orientation 1
(*s*) B3 20 (*s*)_for_1 + Oxy_2 EcoI 3656bp, 1491bp, 18bp 4682bp, 285bp, 18bp Possibly in orientation 1


  1. 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.


Reference Colonies
4/11C 10, 11, 12
4/5I 1, 2, 3
4/6B 2, 3
4/5O 1, 2, 3
2/23N 26, 27, 33
3/20G 28


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


Gene Label Colonies Primers Size Results
(*m*) B1 17, 18, 19, 26 (*m*)_for_1 + Bbs_Methyl_2 1072bp Appears correct
(*s*) B3 20 (*s*)_for_1 + Oxy_2 1220bp Appears correct
(*m*) C5 24, 25 (*m*)_for_1 + (*m*)_rev_1 1225bp 24 possibly too big, 25 appears wrong

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)


[Salicylate] (uM) Abs600
Rep A Rep B Rep C
0 0.512 0.507 0.514
10 0.516 0.516 0.525
100 0.523 0.529 0.528
1000 0.521 0.515 0.518
10000 0.481 0.485 0.484


Time after addition of substrate solution
10 minutes 30 minutes 60 minutes
[Salicylate] (uM) Abs420 Miller units Abs420 Miller units Abs420 Miller units
Rep A Rep B Rep C Rep A Rep B Rep C Rep A Rep B Rep C Rep A Rep B Rep C Rep A Rep B Rep C Rep A Rep B Rep C
0 0.10 0.10 0.12 1005.9 1015.8 1147.9 0.23 0.23 0.23 752.0 752.8 732.8 0.42 0.42 0.42 683.6 695.3 687.4
0 0.12 0.11 0.11 1133.7 1065.9 1085.7 0.24 0.24 0.24 768.7 768.7 749.2 0.45 0.45 0.46 726.6 723.5 715.9
0 0.12 0.12 0.12 1109.0 1115.3 1117.4 0.24 0.25 0.25 783.9 781.3 789.1 0.46 0.48 0.46 734.5 748.3 716.5
0 0.12 0.12 0.11 1151.6 1155.3 1061.8 0.25 0.25 0.22 783.7 812.3 704.6 0.48 0.48 0.49 761.4 771.8 788.3
0 0.12 0.12 0.12 1237.0 1216.5 1239.7 0.26 0.25 0.26 890.5 841.9 898.8 0.48 0.48 0.48 831.6 817.9 824.7


  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!


Label BioBrick ID Enzyme Expected Bands Bands Seen
4/11C p1010 pSB3K3 AvaI 4419bp, 604bp, 569bp, 274bp 604bp
4/5O IS2001 pSB4K5 AvaI, SspI 2044bp, 1546bp, 594bp, 307bp, 18bp 2044bp, 1546bp, 594bp, 307bp
4/6B IS2001 pSB3K5 AvaI, SspI 3153bp, 568bp, 307bp 3151bp, 568bp, 307bp
4/5I IS2001 pSB4A5 AvaI, SspI 1605bp, 1572bp, 987bp, 307bp, 14bp 1605bp, 1572bp, 307bp
4/6B IS2001 pSB3K5 AvaI, NheI 2304bp, 1722bp 2304bp, 1722bp (but not in same digest)


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