Glasgow/Wetlab/Week7

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Week 7

Monday 13th August 2007

  1. Lynsey did colony PCR (see Protocol 9) on the following E. coli TOP10 transformants to check that their plasmids carry our expected inserts:

    Plasmid Expected Insert Primers
    pCR4 Pu Pu_Prefix_After & Pu_Suffix_After
    pCR4 Pu Pu_Prefix_Emma & Pu_Suffix_Emma
    pCR4 Pr Pr_Prefix_1 & Pr_Suffix_1
    pCR4 XylR XylR_Prefix_1 & XylR_Suffix_1
    pCR4 XylR & Pr Pr_Prefix_1 & XylR_Suffix_1


    Overnight cultures were seeded from colonies that gave successful PCR results in order to miniprep the plasmids tomorrow.

  2. Maija did colony PCR (see Protocol 9) on the following E. coli DB3.1 transformants to check that their plasmids carry our expected inserts:

    Plasmid Expected Insert Primers
    pSB3K5 (4/6B) phzM B1 17 phzM_for_1 & BBs_Methyl_2
    pSB1AC3 (3/20G) phzM B1 17 phzM_for_1 & BBs_Methyl_2
    pSB3K5 (4/6B) phzM C5 24 phzM_for_1 & phzM_rev_1
    pSB1AC3 (3/20G) phzM phzM_for_1 & phzM_rev_1
    pSB3K5 (4/6B) DntR DntR_Prefix_1 & DntR_Suffix_2
    pSB1AC3 (3/20G) DntR DntR_Prefix_1 & DntR_Suffix_2


    Overnight cultures were seeded from colonies that gave successful PCR results in order to miniprep the plasmids tomorrow.

  3. Maia took the following BioBricks from the kit plates and transformed (see Protocol 2) into E. coli TOP10 cells:

    BioBrick Part Function Chassis Plasmid Kit Plate Location (Plate/Well)
    [http://partsregistry.org/Part:BBa_B0014 Bba_B0014] Double terminator E. coli TOP10 pSB1AK3 1/1G
    [http://partsregistry.org/Part:BBa_B0015 Bba_B0015] Double terminator E. coli TOP10 pSB1AK3 1/1I
    " " " " 3/3O
    [http://partsregistry.org/Part:BBa_J61100 Bba_J61100] RBS E. coli TOP10 pSB1A2 4/12N
    [http://partsregistry.org/Part:BBa_J61101 Bba_J61101] RBS E. coli TOP10 pSB1A2 4/12J
    [http://partsregistry.org/Part:BBa_J61102 Bba_J61102] RBS E. coli TOP10 pSB1A2 4/12L
    [http://partsregistry.org/Part:BBa_J61103 Bba_J61103] RBS E. coli TOP10 pSB1A2 4/12P
    [http://partsregistry.org/Part:BBa_E0430 Bba_E0430] EYFP + RBS + terminator E. coli TOP10 pSB1A2 1/11A
    [http://partsregistry.org/Part:BBa_E0422 Bba_E0422] ECFP + RBS + terminator + LVA tag E. coli TOP10 pSB1AK3 1/11G
    [http://partsregistry.org/Part:BBa_E0432 Bba_E0432] EYFP + RBS + terminator + LVA tag E. coli TOP10 pSB1A2 4/11C
  4. To ensure successful cloning of the PCR products phzA→phzG, phzA→phzD and phzD→phzG from 10/07, 1ul of GoTaq was added to each 50ul PCR reaction and extended for 10 min at 72°C. Four large lanes of phzA→phzG were run and each had a band of interest of ~8kb . One lane of each phzA→phzD and phzD→phzG was ran and only that of phzA→phzD had a band of interest of ~3kb. Each gel was 1% agarose, 50v for 40 min).
  5. phzA→phzG and phzA→phzD were gel extracted (see Protocol 11 ), cloned into TOPO vectors(see Protocol 12 ), transformed (see Protocol 13 )and grown overnight at 37°C, each reaction divided into 100ul and ~200ul per plate.


Tuesday 14th August 2007

  1. Each plate of transformations of phzA→phzG and phzA→phzD grew several hundred colonies. Eight colonies from plates spread with 100ul of reaction were selected for colony PCR using Reddymix and Touch 2 program (see Protocol 9). Each selected colony was used in a reaction with primers M13_Rev_1 and *F_PstI_For_1, and then with primers M13_For_1 and *F_PstI_For_1. No expected bands were of 2kb were observed in either reaction.
  2. Overnights of Colonies 1 and 2 from each of the above plates (spread with 100ul of transformants) were inoculated overnight in carb LB.
  3. Lynsey took her overnight cultures of Pu and Pr and
  4. Maia did colony PCR (see Protocol 9; program Touch 2) using primers [http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 VR] on the BioBricks we transformed from the kit yesterday, confirming on the basis of product size that our transformants carried the expected plasmids.

Wednesday 15th August 2007

  1. Miniprepped overnights according to QIAGEN manual (see Protocol 5), and were labelled as follows:
    Label 1/1 1/2 2/1 2/2 3/1 3/2 4/1 4/2 5/1 5/2
    Plate 1 1 2 2 3 4 4 5 5
    Colony 1 2 1 2 1 2 1 2 1 2
    Expected Gene phzA→phzG phzA→phzG phzA→phzG phzA→phzG phzA→phzG phzA→phzG phzA→phzG phzA→phzG phzA→phzD phzA→phzD
  2. PCR was done for each of the above minipreps with primers M13_Rev_1 and PhzF_PstI_For_1, and then with primers M13_For_1 and PhzF_PstI_For_1, used Reddymix and Touch2 (see Protocol 9). There is no evidence of a 7 gene operon insert in any of the colonies.
  3. Lynsey repeated amplification of XylR and XylR+Pr with KOD polymerase (see Protocol 9) using the primers listed below and subsequently cloned into TOPO vector (see Protocol 12) and transformed TOP10 cells (see Protocol 13).
    • Primer Pairs:
      • XylR Prefix + XylR Suffix
      • Pr Prefix + XylR Suffix
      • Pr_for_2 + Xylr_rev_2
  4. Maija performed PCR based site-directed mutagenesis for phzM to correct one PstI site and phzS to correct two PstI sites. Only one PstI site can be corrected at the time so Maija started working with both sites separately and proceeding to the second PstI site with the construct where either one has been corrected.
    Biobrick + Construction Vector Primer Pair
    phzM + 3/20G phzM_SDM_PstI_1_for + phzM_SDM_PstI_1_rev
    phzM + 4/6B
    phzS + 3/20G phzS_SDM_PstI_1_for + phzS_SDM_PstI_1_rev
    phzS + 4/6B
    phzS + 3/20G phzS_SDM_PstI_2_for + phzS_SDM_PstI_2_rev
    phzS + 4/6B

    KOD polymerase was used during the mutagenesis PCR, (see Protocol 9) and (seeProtocol 17)

Thursday 16th August 2007

  1. Repeated digests from 9/08 with PstI and EcoRI of minipreps thought to contain phzA→phzG. There is a possibility that colony 4/1 contains the phzA→phzG insert.
  2. Maija did 5 ml overnight cultures with LB kanamycin or LB carbenicillin from the transformed cell colonies 1-18 after the first round of the site-directed mutagenesis in order to get rid of PstI sites inside our becoming BioBrick genes phzM and phzS.
    1. phzM + 4/6B (kanamycin)Site-Directed Mutagenesis
    2. phzM + 4/6B (kan) SDM
    3. phzM + 4/6B (kan) SDM
    4. phzM + 3/20G (carbenicillin) SDM
    5. phzM + 3/20G (carb) SDM
    6. phzM + 3/20G (carb) SDM
    7. phzS + 4/6B (kan) SDM 1. (The first PstI site)
    8. phzS + 4/6B (kan) SDM 1.
    9. phzS + 4/6B (kan) SDM 1.
    10. phzS + 3/20G (carb) SDM 2. (The second PstI site)
    11. phzS + 3/20G (carb) SDM 2.
    12. phzS + 3/20G (carb) SDM 2.
    13. phzS + 4/6B (kan) SDM 2.
    14. phzS + 4/6B (kan) SDM 2.
    15. phzS + 4/6B (kan) SDM 2.
    16. phzS + 3/20G (carb) SDM 1.
    17. phzS + 3/20G (carb) SDM 1.
    18. phzS + 3/20G (carb) SDM 1.

Friday 17th August 2007

  1. Christine did more PCR of the minipreps of the colonies suspected of containing the 7 gene operon using Reddy mix, Touch 2 and the pimer combinations below for each miniprepped colony (see Protocol 9). The result gave no evidence of the insert being present.
  2. Maija miniprepped the mutagenised cell cultures 1-18 and performed a PstI restriction digest with Roche's PstI restriction enzyme to verify whether the mutagenesis worked or not.
  3. On the same 1% agarose gel Maija run the original plasmids which had not been mutagenised but had been digested with the same Roche's PstI restriction enzyme as the mutagenised ones. See the pictures of the gels here. The original plasmids were the following:
    • phzM + 4/6B
    • phzM + 3/20G
    • phzS + 4/6B
    • phzS + 3/20G
  4. From the gel it seems that the phzM minipreps 1., 2., 4. and 5. were successful and thus no PstI site could be seen anymore. These plasmids were sent for sequencing.
  5. The ten phzS minipreps 7.-16. were successful and thus only one PstI site was detected. Maija is going to continue with mutagenising the other PstI site next week.


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