Team:Heidelberg/Templates/Indigoidine week9

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We want to assemble pKH1, which is a pSB1C3-derived plasmid containing pSB1C3-lacPromotor-BBa_B0034-bpsA(pMM64)-

BBa_B0034-svp(pMM65). Single fragments are amplified and cloned using a Gibson approach. We received Streptomyces lavendulae lavendulae DSMXXXX since S. lavendulae ATCC11924 has been shown to carry bpsA

[Takahashi 2007].

Contents

Fragment amplification for pKH1 (Konrad)

  • prepare TB medium for ON (for comparement with LB)
  • colony PCR for plated pMM64 and pMM65 strains from 2013-06-16
  • prepare combinatorial experiment with gibson cloning or CPEC (polysytronic expression of bpsA and svp in TOP10)
  • prepare primer for bpsA only plasmid for BAP1 validation
  • 0.4 µl template
  • 2x 5 µl Primer (1:10 dilution)
  • 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 66 (30x, no touch down) 135
f2: bpsA (AOxA) (NI01,NI02) 68;65 60
f3: bpsA (T) (NI03,NI04)
f4: bpsA (TE) (NI05,NI06)
f5: bpsA (T-TE) (NI03,NI06)
f6: svp (NI07,NI08) pMM65
f7: pSB1C3 (linear.) (NI09,NI10) pSB1C3 with J04450 (pJM03) 66;61 90
Cycles temperature [°C] Time [s]
1 98 30
10 98 1
X1 (incr. down with 0.5 °C) 5
72 Y
20 98 1
X2 5
72 Y
1 72 240
1 4 inf
  • gel purification
180613 delH1aA delH1a bpsa.png

f1: bpsA (all)

180613 indigoidine-f2-6.png

f2-6: single domains of bpsA and svp

180613 indigoidine f7.png

f7: backbone

Assembly of pKH1 and Transformation (Konrad)

Gibson approach

  • molecular ratio = backbone : inserts = 1 : 3
  • partslength: bpsA=3.8 kbp; svp=0.8 kbp; pSB1C3=2.4 kbp
fragment volume [µl] DNA amount [ng]
f2: bpsA (AOxA) 3 126
f5: bpsA (T-TE) 1.5 60
f6: svp 1.5 109
f7: pSB1C3 (linear.) 2 51
total 8
2 µl H2O
10 µl Gibson MM

CPEC approach

Two different approaches were used. In the first approach 3 fragments (backbone (pSB1C3 based), bpsA and svp) were

used for assembly while for the second approach 4 different fragments (Backbone, AOxA, T-TE, svp) were used (like

for the Gibson mix). The amount of used template was calculated for backbone : insert ratio of 1:1 and for insert

fragment ratio at equimolar amount.

  • 3 fragment mix
    • 3 µl template (f7:backbone)
    • 8 µl template (f1:bpsA)
    • 0.3 µl template (f6:svp)
    • 12.5 µl Phusion MM
    • 1.2 µl H2O
  • 4 fragment mix
    • 3 µl template (f7:backbone)
    • 1.7 µl template (f2:AOxA)
    • 1.5 µl template (f5:T-TE)
    • 0.3 µl template (f6:svp)
    • 12.5 µl Phusion MM
    • 6 µl H2O

The used cycler program was carried out similar to CPEC paper for multipart assembly but I forgot to note down the

exact parameters. (Shame on me...)

Transformation

  • into TOP10
  • 4 plates (1:1 and 1:4 of Gibson mix; 1x of each CPEC mix)

Colony PCR

File:180613 gibson-colonyG1-5.png
colony PCR of construct assembeled by Gibson; wanted amplicon size is around 1 kbp

analytical digestion of selected clones

validation of transformed BAP1 with Fussenegger plasmids

fusion PCR

Gibson Assembly of bpsA + svp insert

PCR for colony validation

IPTG induction, titration

IPTG induction, titration RESULT

File:230613-IPTG titration.png
Fussenegger plasmids in BAP1 under various conditions. BAP1 without any Plasmid, BAP1 transformed only with the bpsA carrying plasmid pMM64 and BAP1 transformed with the bpsA carrying plasmid pMM64 and svp carrying pMM65 were induced with four different IPTG concentration ranging from 100 µM to 10 mM at two different temperatures (18 °C and 24°C) for around 24 h.

CPEC and transformation

IPTG induction, titration RESULT

File:240613-IPTG titration.png
2 ml of samples from induction experiement from 2013-06-22 were centrifugated down and then visually compared. Like it can be seen, indigoidine is exported into the supernatant.

Streptomyces cultivation (Ralf)

media for streptomyces culture

  • YEME medium (Kieser et al. 2000)
    • 10 g Glucose
    • 3 g Yeast extract
    • 5 g Bacteriological Peptone (I used Pankreatic Digest of Casein)
    • 3 g Malt Extract
    • 340 g Sucrose
    • ad 1 L water
  • DSMZ medium 65 GYM streptomyces medium
    • 4 g Glucose
    • 4 g Yeast extract
    • 10 g Malt extract
    • (2 g CaCO3 for plates)
    • (12 g Agar for plates)
    • ad 1 L water


Results and Discussion

BAP1+pMM64+pMM65 eventually produced indigoidine. We will now focus on assembling our own constructs starting with

pKH1. Streptomyces cultures look like yeast and have a strong smell as in the forest.

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