Team:Heidelberg/Templates/MM week17p

From 2013.igem.org

Contents

2013-08-19

  • received BL21(DE3) from Gruss Lab, ZMBH
  • grow in LB to OD=0.53, prepare competent cells
  • co-transform with pIK1.2(0.2 µl = 120 ng)+pMM64(1 µl = 15 ng), pIK2.6(0.2 µl = 60 ng)+pMM64(1 µl = 15 ng), plate on Cm+Amp+IPTG, grow at 37°C

2013-08-20

  • all colonies white
  • pick 1 colony each, inoculate LB+Cm+Amp, grow at 37°C until cultures are opaque, add IPTG to 1mM, grow at 30°C
  • no visible indigoidine production
  • need to clone into a different backbone in order to be able to cotransform with pRB21
  • inoculate LB+Kan with TOP10-pSB3K3-BBa_J04450, grow at 37°C

2013-08-21

Lane 1: NEB 2-log; lanes 2-3: pSB3K3-BBa_J04450 digested with EcoRI+SpeI; lane 4: pIK1.2 digested with EcoRI+SpeI; lane 5: pIK2.6 digested with EcoRI+SpeI
  • prepare glycerol stock of TOP10-pSB3K3-BBa_J04450
  • perform miniPrep -> 10.1 ng/µl in 38 µl
  • digest with EcoRI+SpeI: 380 ng of pSB3K3-BBa_J04450: use everything (50 µl total volume, 2x0.5 µl enzyme, expect 2.7kb & 1kb), 1200 ng of pIK1.2 (2 µl of miniPrep from 2013-08-09, 20 µl total volume, 2x0.5 µl enzyme, expect 6.7kb & 2.7kb), 900 ng of pIK2.6 (3 µl of miniPrep from 2013-08-09, 20 µl total volume, 2x0.5 µl enzyme, expect 4.4kb & 2.7kb)
  • pSB3K3-BBa_J04450 looks as though it was not cut: inoculate 50 ml TB+Kan with TOP10-pSB3K3-BBa_J04450, grow at 37°C
  • as control for indigoidine production from pMM64: co-transform BL21(DE3) with pMM64(1 µl = 15 ng)+pMM65(0.2 µl = 13 ng), plate on Amp+Kan+IPTG

2013-08-22

Lane 1: pSB3K3-BBa_J04450 digested with EcoRI+SpeI, lane 2: NEB 2-log
Lane 1: NEB 2-log; lanes 2-9: pRB* plasmids; lane 10: 1 µl of pSB3K3-BBa_J04450 midiPrep
  • no colonies of BL21(DE3)-pMM64-pMM65: co-transform BL21(DE3) with pMM64(1 µl = 15 ng)+pMM65(1 µl = 75 ng), plate on Amp+Kan+IPTG
  • prepare midiPrep of pSB3K3-BBa_J04450: 2952.1 ng/µl
  • digest 0.4 µl of midiPrep with EcoRI+SpeI (20 µl total volume, 2x0.5 µl enzyme)
  • band too weak: 2 possibilities:
    1. concentration measurement wrong
    2. mispipeted
  • => load 1 µl of midiPrep on gel -> concentration measurement about right
  • large pSB3K3 fragment over 3 kb, consistent with miniPrep from 2013-08-21 -> 2.7 kb expected, sequence wrong on parts.igem.org
  • gel-purify pSB3K3, 6 kb fragment of pIK1, 4 kb fragment of pIK2
  • 18 µl of pSB3K3 (not measured), 12.3 ng/µl pIK1 in 21 µl; 7.9 ng/µl pIK2 in 21 µl
  • ligate at RT for 1h:
what µl
pSB3K3 9
insert 8
T4 ligase 1 µl
T4 ligase buffer 2 µl
  • heat-inactivate: 75°C for 5 min
  • transform 10 µl of each ligation into TOP10, plate on Kan, grow at 37°C (pSB3K3+pIK2 = pIK6)

2013-08-23

Colony-PCR of TOP10 transformed with pSB3K3 ligated with pIK1/pIK2 and primers RB43+VR. Lane 1: NEB 2-log; lanes 2-6: pIK1; lanes 7-11: pIK2
Colony-PCR of TOP10 transformed with pSB3K3 ligated with pIK2 and primers RB43+VR. Lane 1: NEB 2-log; lanes 2-11: pIK2
  • pick 5 colonies each, run colony-PCR with primers RB43+VR (expected: 1kb, using iTaq, 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
  • no amplificate
  • have another look at sequences (motivated by DelRest results): 1 bp missing in pIK1.2: frame shift in permeability device => send pIK1.3 to sequencing with primers VF2, VR, IK25
  • pick 30 colonies of pSB3K3-pIK2, run colony-PCR with primers RB43+VR (pool 3 colonies in 1 PCR reaction, iTaq, 20 µl total volume)
  • no positives => ligation did not work, most likely due to low DNA concentrations
  • need to repeat: inoculate 2x5 ml 2xYT+Cm with TOP10-pIK2.6

2013-08-24

Lane 1: NEB 2-log; lane 3: pSB3K3 digested with EcoRI+SpeI; lane 5: pIK2.6 digested with EcoRI+SpeI
  • make miniPrep of pIK2.6: 97 ng/µl in 37.5 µl
  • digest 3 µg pSB3K3 (1 µl of midiPrep from 2013-08-22), 1.7 µg pIK2.6 (17 µl of miniPrep) with EcoRI+SpeI (20 µl total volume, 0.5 µl of each enzyme)
  • treat pSB3K3 digest with antarctic phosphatase (37°C, 60 min)
  • run gel, gel-purify -> 18 µl pSB3K3 (not measured), 15.0 ng/µl pIK2.6 in 20.5 µl
  • ligate at RT for 1h:
what µl
pSB3K3 9
pIK2.6 8
T4 ligase 1 µl
T4 ligase buffer 2 µl
  • heat-inactivate: 75°C for 5 min
  • transform 10 µl of ligation into TOP10, plate on Kan, grow at 37°C

2013-08-25

Colony-PCR of TOP10 transformed with pSB3K3 ligated with pIK2 and primers RB43+VR. Lane 1: NEB 2-log; lanes 2-11: pIK2
  • pick 10 colonies, run colony-PCR with primers RB43+VR (expected: 1kb, using iTaq, 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
  • no amplificate
  • VR primer might not be binding to pSB3K3: http://www.ccbi.cam.ac.uk/iGEM2006/index.php/Primer_List#BioBrick_verification_primers , https://2009.igem.org/Team:Groningen/Notebook/22_July_2009
  • pick another 10 colonies, run colony-PCR with primers VF2+IK25 (expected: 1kb, iTaq, 20 µl total volume):
Colony-PCR of TOP10 transformed with pSB3K3 ligated with pIK2 and primers VF2+IK25. Lane 1: NEB 2-log; lanes 2-11: pIK2
Cycles temperature [°C] Time [s]
1 95 300
35 95 30
54 30
72 90
1 72 600
1 10 inf
  • definite product, but at 1.3 kb => pSB3K3 was longer than expected in digest, might be a result from that
  • unspecific bands probably due to melting temperature of IK25 being at 67°C
  • inoculate 2xYT+Kan, grow at 37°C
  • pSB3K3 used was from plate 5 => transform TOP10 with pSB3K3 from plate 2, well 6 F
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