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Week 6

Monday 6th August 2007

  1. Maia checked the presence and size of phzM and phzS inserts within the Topo vector by repeating the PCR performed on 02/08. Results confirmed presence of inserts with the correct size. The TOPO vectors containing phzM and phzS were then sent for sequencing to ensure the inserts did not contain any mistakes. DNA sequenced and the primers used are summarized in the table below.
    Label B1 B3 C5
    Gene phzM phzS phzM
    Colony 17,18,19,26 20 25,24
    Primers phzM_for_1 + Methyl _2 phzS_for_1 + Oxy_2 phzM_for_1 + phzM_rev_1

    (NB - For each 2 x 10µl was sent)

  2. Christine repeated PCR of the 7 gene operon and 2x 3.5kb gene operons (phzA→phzD and phzD→phzG) with the intention to obtain a large amount of PCR product. PCR performed was a repeat of the PCR from 31/07, with annealing temperature of 58°C.

Tuesday 7th August 2007

  1. Christine transformed TOP10 cells with TOPO vector containing the 7 gene operon. 4 µl of PCR product was cloned into TOPO vector. Only 2 colonies of (phzA→phzD) grew, and no (phzA→phzG)grew. (see Protocol 12)
  2. (phzD→phzG) was amplified again with KOD XL and KOD using the respective programs (see Protocol 9). Primers used: phzD_for_1 and phzG_rev_1 .Expected size is 3.5 kb.

Wednesday 8th August 2007

  1. A digest scheme was designed to allow for the insertion of our biobricks into construction vectors.
    Biobricks : DntR, phzS, phzM, XylR, XylR + Pr, Pr, Pu
    Construction Vectors: 3/20G, 4/6B, 4/50
  2. phzD→phzG was gell extracted, cloned into TOPO vectors and transformed into TOP10 cells. (see Protocol 12). No phzD→phzG transformations grew.
  3. Overnights were set up for phzA→phzG in carbencilln LB.

Thursday 9th August 2007

  1. Each biobrick and construction vector was digested with EcoRI and SpeI (Roche)(see Protocol 7)
  2. Digest products were resolved on 1% agarose gels and either gel extracted (see Protocol 11) or PCR purified (see Protocol 14)
  3. Each biobrick was ligated with three different construction vectors (listed above) using the Quick Ligation Kit (New England Biolabs) (see Protocol 15)
  4. Ligation products were transformed into TOP10 cells and grown on LB agar plates supplemented with appropriate antibiotic.
  5. Overnights of both phzA→phzG colonies were miniprepped according to QIAGEN miniprep kit protocol (see Protocol 5).
  6. PCR was carried out on the phzA→phzG minipreps using KOD XL and KOD XL program with annealing temperature of 58°C (see Protocol 9). Primers used were Bbp_phzA_for_1 and Bbs_phzG_rev_1. No amplicons were observed.
  7. Restriction digests were set up for phzA→phzG according to Roche Protocol using Buffer H (see Protocol 7). Expected fragment sizes were not observed.
    phzA→phzG Colony Enzyme Fragment Sizes in Orientation 1 Fragment Sizes in Orientation 2
    1 + 2 EcoRI 4582bp, 2622bp, 2997bp, 18bp 6367bp, 2622bp, 18bp, 1212bp
    1 + 2 PstI 7789bp, 486bp, 1451bp, 494bp 4380bp, 486bp, 1451bp, 3902bp

Friday 10th August 2007

  1. Results of construction vector transformations: (number of colonies per plate)
    Construction Vector
    Biobrick 3/20G 4/50 4/6B
    Pu 0 2 3
    Pr 0 V.small V.small
    XylR 2 3 V.small
    XylR + Pr 0 0 0
    DntR 5 3 3
    phzM 12 V.small 8
    phzS 31 1 56
  2. Maija did a colony PCR from six plates to make sure the inserted genes are in the right constructor vectors. The primers were chosen so that one of the primers anneals to the insert and the other to the vector. As a result the colonies phzM, phzS and DntR gave the right size bands when run on an 1% agarose gel. Pu colony failed to give any PCR product. See the pictures of the gels here.
    Plasmid Expected Insert Primers
    4/6B Pu Pu_Prefix_2 & VR and
    VF2 & Pu_Suffix_2
    4/6B DntR VF2 & DntR_Suffix_2 and
    DntR_Prefix_1 & VR
    3/20G phzM VF2 & phzM_rev_1 and
    VF2 & Methyl_2
    4/6B phzM VF2 & phzM_rev_1 and
    VF2 & Methyl_2
    3/20G phzS VF2 & Oxy_2 and
    Oxy_1 & VR
    4/6B phzS VF2 & Oxy_2 and
    Oxy_1 & VR
  3. PCR redone in multiple (repeated from 31/07) to attain large amounts of DNA for phzA→phzG using KOD XL and KOD program, and phzA→phzD and phzD→phzG using KOD and KOD program - both with annealing temperature of 58°C (see Protocol 9).


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