Team:Heidelberg/Templates/MM week21p

From 2013.igem.org

Contents

2013-09-16

  • send pIK8.6, pIK8.9 to sequencing with primer RB43

2013-09-17

2013-09-18

Lane 1: NEB 2-log; lane 2: pIK8.6 digested with EcoRI+SpeI, lane 3: pSB1C3 digested with EcoRI+SpeI
  • sequences arrived: PIK8.6-pIK8.9-2013-09-18.zip, PIK8.6-2013-09-18 VR.clustal.txt, File:Heidelberg_PIK8.9-2013-09-18 VR.clustal.txt
  • sequences look good
  • make miniPrep of pIK8.6 -> 269 ng/µl in 38 µl
  • to verify functionality of permeability device: need to co-transform with indigoidine synthetase => need to clone into pSB3K3 -> pIK9
  • need to clone into pSB1C3 for submission (pIK10, pIK11): digest 465 ng pSB1C3 (1 µl of Ralf's midiPrep) with EcoRI+SpeI, treat with antarctic phosphatase (37°C, 60 min)
  • digest pIK8.6 with EcoRI+SpeI (3 µl DNA, 0.5 µl enzyme, 20 µl total volume)
  • gel-purify pIK8.6 insert (20 ng / µl in 20 µl), pSB1C3 (18 µl, concentration not measured)
  • ligate for 1 h at RT:
pIK9 pIK10 pIK11
backbone 9 µl pSB3K3 ES from 2013-09-27 9 µl pSB1C3 ES 9 µl pSB1C3 ES
insert 8 µl pIK8.6 ES 8 µl pIK8.6 ES 4.5 µl pIK2.6 ES (rest from 2013-08-24)
T4 ligase 1 µl 1 µl 1 µl
T4 ligase buffer 2 µl 2 µl 2 µl
H2O 0 µl 0 µl 3.5 µl
  • transform TOP10 with 10 µl of each ligation, plate on LB+Kan(pIK9), LB+Cm(pIK10, pIK11)

2013-09-19

Lane 1: NEB 2-log; lanes 2-5: pIK9; lanes 6-9: pIK10; lanes 10-13: pIK11
  • colonies present for all ligations
  • colonies for pIK10 extremely small -> permeability device toxic
  • run colony-PCR using primers VF2+IK25 (expected: 1 kb, OneTaq, 20 µl total volume):
Cycles temperature [°C] Time [s]
1 95 300
35 95 30
54 30
72 90
1 72 600
1 10 inf
  • grow in TB+Kan (pIK9) / TB+Cm (pIK10, pIK11)
  • evening: make miniPreps of pIK9, pIK11 (pIK10 grew very slowly) -> ca. 50-60 ng/µl (pIK9), 100-300 ng / µl (pIK11) in 38 µl
  • inoculate 2xYT+Cm with pIK8.1, pIK8.6

2013-09-20

  • make miniPreps of pIK10 -> 400-600 ng/µl in 38 µl
  • digest everything with BamHI+HindIII, expected: 2.1 kb + 3.1 kb + 4.1 kb (pIK9), 2.1 kb + 6.6 kb (pIK10), 2.1 kb + 4.3 kb (pIK11) (20 µl total volume, 0.5 µl enzyme, using 0.5 µl (pIK10), 1 µl (pIK11), 2 µl (pIK9) DNA)
  • to distinguish between pSB3C5 and pSB1C3: digest pIK10, pIK11 with XhoI (expected: 1 band for pSB3C5, 3 bands for pSB1C3)
  • send pIK10.1, pIK10.3, pIK10.3, pIK11.1, pIK11.2 to sequencing with primers VF2, VR
  • test permeability device:
    • inoculate 3 ml LB+Cm soft agar with 100 µl of pIK8.1, pIK8.6 ON culture, pour on LB+Cm plates
    • add sterile filter paper, pipet 8 µl, 4 µl, 2 µl, 1 µl of Bacitracin on filter paper discs
    • grow at 37°C
Left: TOP10-pIK8.6, right: TOP10-pIK8.1 (negative control)
counterclockwise, starting top right: 8 µl, 4 µl, 2 µl, 1 µl bacitracin
  • Co-transform TOP10 with 25 ng pIK9.1, 10 ng pRB22.4, plate on Cm+Kan, grow at 37°C

2013-09-21

  • co-transformats: lots of extremely small, blue-ish colonies, several big white colonies -> possibly mutated indigoidine synthetase, several big black colonies -> possibly mutated permeability device
  • pick one little blue-ish colony, grow in 2xYT+Cm+Kan at 37°C