Team:Heidelberg/Templates/Indigoidine week10

From 2013.igem.org


In parallel to the experiments with the pMM-plasmids (Konrad) we start to get the native bpsA from Streptomyces

lavendulae lavendulae DSMXXXX (Ralf).

Contents

Indigoidine production with pKH1 (Konrad)

colony PCR

transformation

analytic digestion

fragment amplification

PCR for bpsA and backbone amplification and Phusion polymerase validation (lanes to the right side of ladder; white stripe on the right is not a band); run at 135 V, 0.8 % gel (TAE); wanted amplicon size are: (bpsA: ~3.8 kbp; linearized pSB1C3: ~2.4 kbp)

==> for two fragments with our Phusion MM and Phusion MM of SYNtheSYS (old, don't know if functional...)

  • 0.4 µl template
  • 2x 0.5 µl Primer
  • 25 µl Phusion MM
  • 14.6 µl H2O
fragment primer template (DNA) annealing temp (X1;X2) [°C] elongation time (Y) [s]
f1: bpsA (all) (NI01,NI06) pMM64 65;66 120
f7: pSB1C3 (linear.) (NI09,NI10) pSB1C3 with J04450 (pJM03) 66;61 75
Cycles temperature [°C] Time [s]
1 98 30
10 98 5
X1 (incr. down with 0.5 °C) 15
72 Y
20 98 5
X2 15
72 Y
1 72 360
1 4 inf
  • RESULT: no PCR product for both amplicons, maybe due to wrong cycle conditions


construction of pSB1C3-bpsA-svp pRB1

Streptomyces lavendulae lavendulae DSMZXXXX was cultivated in GYM medium from freeze dried cell pellet at 28 °C and 170 rpm. cultivation of S. lavendulae lavendulae

  • freeze dried cell pellet in 5 ml medium 65
  • 3x 200 ul in 3 mL medium 65 and YEME, respectively. 28 °C 200 rpm (3 p.m.)
  • 3x agar plate medium 65, 50/75/100 ul inoculation; 28 °C (7 p.m.)
  • prepare YEME agar for next day

PCR with Gibson Primers RB05-RB10

  • pSB1C3 backbone from PCR product 50 uL Phusion Flash; 05 uM Primer, 25 uL Master mix, 19,4 uL water, 0,6 uL pSB1C3
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 40
1 72 420
1 4 inf Gibson assembly and
  • BpsA colony PCR 50 ul Phusion Flash; 0,5 uM Primer, 25 uL Master Mix, 20 uL colony with water
  • colony was picked and held into liquid nitrogen/ warm water repeatedly
Cycles temperature [°C] Time [s]
1 98 11
1 65 5
1 72 60
30 98 1
72 60
1 72 300
1 4 inf
  • pMM65 PCR for svp 50 ul Phusion Flash; 0,5 uM Primer, 25 uL Master mix, 15 uL water, 5 uL 23 ng/ul pMM65
Cycles temperature [°C] Time [s]
1 98 11
1 65 5
1 72 15
30 98 1
72 15
1 72 300
1 4 inf

Agarose Gel

gel with 17 ul PCR product, 3 ul 6x buffer, 6 ul 2-log

  • pSB1C3 backbone
    • weak band on gel < 50 ng/ 17 ul
    • Troubleshooting
      • two step PCR -> try annealing step with 66 °C annealing temp (NEB Tm Calculator Phusion)
      • circular template
      • template sequence?
  • bpsA
    • cells not lysed? DNA damaged (nitrogen)?
  • svp

#2 PCR pSB1C3 RB09/10

  • 8 ul water
  • 10 ul Phusion Flash HF MM
  • je 0,5 ul Primer 100 uM
  • 1 ul pSB1C3 1:5
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
62 5
72 45
1 72 180
1 4 inf

PCR svp RB07/08 from pMM65 23 ng/ ul circular

  • 8 ul water
  • 10 ul Phusion Flash HF MM
  • je 0,5 ul Primer 100 uM
  • 1 ul pMM65 1:10
Cycles temperature [°C] Time [s]
1 98 15
30 98 1
62 5
72 15
1 72 300
1 4 inf

PCR bpsA RB05/06 from S. lavendulae

  • Streptomyces was washed 3x in H20 dest; 2 "colonies" in 1 uL
  • 8 ul water
  • 10 ul Phusion Flash HF MM
  • je 0,5 ul Primer 100 uM
  • 1 ul Streptomyces culture
Cycles temperature [°C] Time [s]
1 98 180
30 98 1
62 5
72 60
1 72 300
1 4 inf

0,8 % Agarose Gel

2nd run PCR for RB construct 1
  • pSB1C3 worked well with 20-50 ng/ ul
  • BpsA
    • annealing temp still too high?
    • cell lysate destroys master mix? -> nitrogen/ boil before PCR, use liquid phase
    • cells not lysed? vortex with glas beads

#3 desperation PCR

PCR water Phusion Flash HF MM Primer 100 uM DMSO template template
Ia610RB05, RB06 à 0,5 ul12S. lav, medium 65, nitrogen shock, glass beads vortex, 98 °C with water

bidest; liquid phase

Ib610RB05, RB06 à 0,5 ul03S. lav, medium 65, nitrogen shock, glass beads vortex, 98 °C with water

bidest; liquid phase

II610RB05, RB06 à 0,5 ul03S. lav, medium 65, glass beads vortex, 98 °C with water bidest; liquid phase
III610RB05, RB06 à 0,5 ul03S. lav, medium 65, nitrogen shock, 98 °C with water bidest; liquid phase
IVa610RB05, RB06 à 0,5 ul12S. lav, medium 65, 98 °C with water bidest; liquid phase
IVb610RB05, RB06 à 0,5 ul03S. lav, medium 65, 98 °C with water bidest; liquid phase
V610RB05, RB06 à 0,5 ul03S. lav, medium 65, nitrogen shock, glass beads vortex; pellet
VI610RB05, RB06 à 0,5 ul03S. lav, YEME medium, nitrogen shock, glass beads vortex; pellet/ liquid

phase

VII610RB05, RB06 à 0,5 ul03S. lav, YEME medium, nitrogen shock, 98 °C with water bidest; liquid phase
VIII710RB07, RB08 à 0,5 ul11pMM65 2,3 ng/ ul, trace RB05
IX610RB07, RB08 à 0,5 ul01pMM65 2,3 ng/ ul, should be 8 ul water
Cycles temperature [°C] Time [s]
1 98 120
2 98 1
40 5
72 70
2 98 1
45 5
72 70
5 98 1
50 5
72 70
5 98 1
55 5
72 70
15 98 1
65 5
72 70
1 72 300
1 4 inf

Desperation Gel

0,8 % Agarose, 100 V, 60 min.
  • band around 200 bp? unspecific product due to PCR conditions, but then why no product?
  • VI glowing pocket -> sucrose in YEME medium? -> PCR purification and new gel -> still nothing

Phusion Flash HF has proofreading activity; so mutations may stop elongation -> try intron iTaq and design new

primers without mutations.


  • PCR svp from NI7/8 PCR product and pMM65, respectively; svp with NI7/8 as control; intron iTaq 2x Master mix.
PCRiTaq 2x Master MixtemplatePrimer 10 uMwater
A10 ul pMM65 2 ul 2,3 ng/ ul 1 ul RB07 and RB08 6 ul
B10 ul pMM65 2 ul 2,3 ng/ ul 2 ul RB07 and RB084 ul
C10 ul svp 2 ul 7,2 ng/ ul 1 ul RB07 and RB08 6 ul
D10 ul svp 2 ul 7,2 ng/ ul 2 ul RB07 and RB084 ul
E10 ul svp 1 ul 7,2 ng/ ul 1 ul NI07 and NI088 ul
Cycles temperature [°C] Time [s]
1 94 120
35 94 20
60 10
70 60
1 72 240
1 4 inf

Gel 0,8 % 100 V 60 min

PCR with RB-Primers and intron iTaq
  • nothing
    • intron iTaq maybe doesn't work so well since control isn't working; or standard protocol is bad.
    • run PCR with other Taq Master Mix
  • iTaq with Slav colony YEME
TubeMaster MixMM 2x [ul]Primer 100 mM [ul]templatetemplate [ul]water [ul]
A:svp RB7/8Fermentas Taq PCR Master Mix 2x257,5svp 7,2 ng/ ul100
B:svp RB7/8Fermentas Taq PCR Master Mix 2x257,5svp 7,2 ng/ ul100
C:pMM65 RB7/8Fermentas Taq PCR Master Mix 2x257,5pMM65 2,3 ng/ ul100
D:pMM65 RB7/8Fermentas Taq PCR Master Mix 2x257,5pMM65 2,3 ng/ ul100
E:pMM65 NI7/8Fermentas Taq PCR Master Mix 2x257,5pMM65 2,3 ng/ ul100
F:svp RB7/8Finnzymes Taq PCR Master Mix 2x257,5svp 7,2 ng/ ul100
G:svp RB7/8Finnzymes Taq PCR Master Mix 2x257,5svp 7,2 ng/ ul100
H:pMM65 RB7/8Finnzymes Taq PCR Master Mix 2x257,5pMM65 2,3 ng/ ul100
I:pMM65 RB7/8Finnzymes Taq PCR Master Mix 2x257,5pMM65 2,3 ng/ ul100
J:pMM65 NI7/8Finnzymes Taq PCR Master Mix 2x257,5pMM65 2,3 ng/ ul100
K:svp RB7/8NEB OneTaq Quick Load PCR Master Mix 2x257,5svp 7,2 ng/ ul100
L:svp RB7/8NEB OneTaq Quick Load PCR Master Mix 2x257,5svp 7,2 ng/ ul100
M:pMM65 RB7/8NEB OneTaq Quick Load PCR Master Mix 2x257,5pMM65 2,3 ng/ ul100
N:pMM65 RB7/8NEB OneTaq Quick Load PCR Master Mix 2x257,5pMM65 2,3 ng/ ul100
O:pMM65 NI7/8NEB OneTaq Quick Load PCR Master Mix 2x257,5pMM65 2,3 ng/ ul100

500px|2-log; A-E; 2-log; F-J

2-log; K-O; 2-log; Slav IVb; VI; V; VII

  • gel analysis
    • svp worked well as template for RB07/08, but lots of wrong product around 100-150 bp. Maybe primers bind to each other, folding results in other preferred sequence or gibson overlap has big affinity though alignments didn't show that
    • pMM65 amplification only with NEB OneTaq
    • no product with NI07/08 primers...because they are for TE-domain of BpsA. Ni09/10 would have been the right choice
    • for further runs, use OneTaq and Fermentas Taq
    • was svp amplified or is it just template?
    • Again glowing pocket in Slav VI
    • huge amount of primers was used in al reactions

PCR with Phusion Flash HF from PCR products (yesterday with Taq-polymerases)

  • 10 ul MM; Primer je 2 ul unv.; water 4 ul; template 2 ul
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
55 5
72 30
1 72 300
1 4 inf

gel 9 ul 1x loading buffer, 1 ul pcr product

2-log / A / A-Phu Fl / F / F-Phu-Fl / K / K-Phu-Fl / svp 7,2 ng template
  • gel analysis
    • no significant amplification of svp with Phusion Flash
    • Taq PCR amplified template
    • run different protocol
  • PCR Phusion flash taq-amplified svp 2
    • 10 ul MM, 1 ul primer, 7 ul water, 1 ul water
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 30
1 72 180
1 4 inf
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 35
1 72 180
1 4 inf

gel with 9 ul 1x loading buffer and 1 ul pcr product

2-log / wrong / wrong / 3-step K / 3-step K-Phu-Fl / 2-step K / 2-step K-Phu-Fl / K template / 2-log
  • gel analysis
    • 3-step worked better than 2-step
    • primers might prime each other or the fragment
    • it's not just too much primer because lane 4/5 are a lot brighter than 6/7 -> the 120 bp-band is an

amplification product. -> order S. verticillus and published primers from Takahashi and Sanchez -> order published Primers for sfp, entD (Lambalot), bpsA (Takahashi) -> order stuff for DNA isolation of Streptomyces

Results and Discussion

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