Team:Heidelberg/Templates/Indigoidine week10

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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

File:2013-07-02 DelHf1a DelHf2 M bpsA pSB1C3.jpg
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
20130706 svp6 1.PNG

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

20130706 svp6 2.PNG

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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