Supporting Information
Efficient Production of Single-Stranded Phage
DNA as Scaffolds for DNA Origami
Benjamin Kicka, Florian Praetoriusb, Hendrik Dietzb, Dirk Weuster-Botz*a
[a]
Institute of Biochemical Engineering, Technische Universität München,
Boltzmannstr. 15, 85748 Garching, Germany
[b]
Physik Department, Walter Schottky Institute, Technische Universität München,
Am Coulombwall 4a, 85748 Garching, Germany
S1
Host strain and phage variants
As host strain Escherichia coli XL1-Blue MRF’ Kan (Δ(mcrA)183 Δ(mcrCB-hsdSMRmrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F ́ proAB lacIqZΔM15 Tn5 (Kanr)])
(Stratagene) was used. The bacterial strain was stored in medium with 15 % glycerol at
-80 °C.
Phage variants with different ssDNA lengths were produced using heat shock
transformation of chemically competent cells with ssDNA (7249, 7560, 8064 bases).1 1 µL
100 nM ssDNA was added to 90 µL chemical competent cells and incubated for 30 min on
ice. Heat shock transformation was performed for 30 s at 42 °C followed by 5 min
incubation on ice. The transformed cells were incubated for 30 min with 300 µL Lysogeny
Broth (LB) media at 37 °C, shaking at 150 rpm. 200 µL transformed cells were plated on
prewarmed LB plates containing 50 mg L-1 Kanamycin A. Plaques were detectable after
incubation at 37 °C over night (14 – 20 h). With a sterile pipette one plaque was touched
and growing XL1-Blue cells at an optical density of 0.4 in 50 mL LB containing Kanamycin
A were infected. Cultivation of infected cells at 37 °C and 250 rpm in a shaking flask for
5 h lead to phage titer up to 2 ∙ 1011 pfu mL-1. The host cells were separated for 20 min at
3260 rcf and the supernatant was used for infection of the fed-batch cultivation in the
stirred-tank reactor.
Preculture preparation
The preculture was produced in defined mineral medium based on Riesenberg with
20 % (v/v) inoculation volume and was cultivated for 6.5 h at 250 rpm, 37 °C in shake
flasks.2 The defined media contains following components: mineral media in g L-1:
13.3 KH2PO4, 4 (NH4)2HPO4, 1.7 citric acid; trace elements in mg L-1: 2.5 CoCl2 · 6 H2O,
15 MnCl2 · 4 H2O, 1.5 CuCl2 · 2 H2O, 3 H3BO3, 8.4 EDTA 2 Na · 2 H2O, 60 Fe(III)-citrate,
2.5 Na2MoO4 · 2 H2O, 8 Zn(CH3COO)2 · 2 H2O; the following amount of sterile additives
were added: 5 g L-1 Glucose, 1.2 g L-1 MgSO4 · 7 H2O, 1 mM Thiamin-HCl (sterile filtered).
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Feed profile
The predefined feed profile for exponential growth was calculated from following equation.
𝑉̇ =
1
𝑐𝑆0
µ
(𝑌 𝑠𝑒𝑡 ) 𝑐𝑋0 𝑒 µ𝑠𝑒𝑡 (𝑡−𝑡0 ) 𝑉
𝑋𝑆,µ
µset
…
predefined growth rate, h-1
cS0
…
substrate concentration feed, g L-1
cX0
…
biomass concentration at t0, g L-1
t
…
time, h
V
…
reaction volume at t0, L
𝑉̇
…
volumetric flow rate, L h-1
YXS,µ
…
biomass yield, gCDW gGlc-1
Equation 1
The volume specific mass flow (Fig. 1a) was calculated from the glucose concentration of
the feed solution and the actual reaction volume. The biomass at the end of the batch was
10.8 g L-1 and the biomass yield was 0.43 gCDW gGlc-1 for feed equation.
Shake flask based fermentation
Escherichia coli XL1-Blue MRF’ Kan cultures were grown in EnPresso B medium
(BioSilta, Camebridgeshire, UK) according to the manufacturers protocol. Phage infection
and downstream processing was performed as described above.
Downstream processing
Host cells were centrifuged after harvest for 1 h at 20 °C and 3260 rcf (Rotixa 50 RS,
Hettich AG, Bäch, Switzerland). The bacteriophages in the supernatant were precipitated
with 2.5 % (m/v) Polyethylene glycol 8000 and 3 % (m/v) NaCl for 1 h at RT and 800 rpm.
After separation at 3260 rcf and 20 °C for 30 min the precipitated phage pellet was
resuspended in 200 mL TE buffer (10 mM Tris HCl, 1 mM EDTA, pH = 8.5).
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Scheme S1. Downstream processing of ssDNA including following steps: separation of
host cells and extracellular produced bacteriophages, polyethylene glycol mediated phage
precipitation, chemically phage lysis and ssDNA precipitation with ethanol.
phage
precipitation
phage lysis
(chemical)
host cells
1st DNA precipitation
50 % EtOH (v/v)
2nd DNA precipitation
75 % EtOH (v/v)
ssDNA
centrifugation stirred reaction system reaction vessel
The phages were lysed adding 400 mL lysis buffer (Qiagen Buffer P2, Cat. No. 19052,
200 mM NaOH, 1 % SDS) and 300 mL neutralization buffer (Qiagen Buffer P3, Cat. No.
19053, 3 M potassium acetate, pH = 5.5) and incubated on ice for 15 min. Centrifugation
was performed afterwards for 30 min, at 3260 rcf and 4 °C prior to ssDNA precipitation
with 50 % (v/v) chilled ethanol and incubated for at least 1 h on ice. The precipitated
ssDNA was separated for 10 min at 3260 rcf and 4 °C. In a secondary ethanol
precipitation step the ssDNA pellet was washed with 75 % chilled ethanol for 15 min on
ice and centrifugated for 10 min at 13.000 rcf (Heraeus Biofuge Stratos, Thermo Fisher
Scientific Inc., Waltham, USA). The ssDNA pellet is formulated in TE buffer at a final
concentration of 1 µM and stored at -20 °C.
Analytics
The growth of the host cells was measured atline using optical density at 600 nm
(Genesys 10S UV-VIS, Thermo Fisher Scientific Inc., Waltham, USA) and offline with the
determination of cell dry weight in triplicates. The concentrations of glucose and ammonia
were determined using enzymatic assays (D-Glucose: Kit No: 10 716 251 035,
Ammonium: Kit No: 11 112 732 035, Boehringer Mannheim/R-Biopharm, Darmstadt,
Germany). The enumeration of bacteriophages in the supernatant after host cell
separation for 10 min (13.000 rcf, 25 °C) was determined using Direct Plating Plaque
Assay.3 Quantification of bacteriophages in ultraviolet light is based on a recently
published method, measuring the absorbance of phage protein and ssDNA at 269 nm.
4
Prior to analysis, the bacteriophages from 1.5 mL supernatant were precipitated by PEG
based precipitation (40 g L-1 PEG 8000, 30 g L-1 NaCl, 30 min, 25 °C) and resuspended in
TE buffer for measurement. The concentration of purified ssDNA was calculated using
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absorbance at 260 nm and the respective extinction coefficient (ssDNA 7249: 7.12 107 M1
cm-1, 7560: 7.43 107 M-1 cm-1, 8064: 7.91 107 M-1 cm-1).
Agarose gel electrophoresis was performed in 2 % agarose gels (UltraPure Agarose,
Invitrogen) containing electrophoresis buffer (1 mM EDTA, 44.5 mM Tris base, 44.5 mM
boric acid, and 11 mM MgCl2, pH 8.4) and 0.4 µg mL-1 ethidium bromide. Samples were
electrophoresed for 90 min at 90 V in a water bath. Gels were imaged using a Typhoon
9500 FLA laser scanner (GE Healthcare) at a resolution of 25 µm px-1 (excitation at 535
nm, emission > 575 nm).
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References
1. Douglas, S. M.; Dietz, H.; Liedl, T.; Högberg, B.; Graf, F.; Shih, W. M. Nature 2009, 459
(7245), 414–418, DOI:10.1038/nature08016.
2. Riesenberg, D.; Schulz, V.; Knorre, W. A.; Pohl, H. D.; Korz, D.; Sanders, E. A.; Roß,
A.; Deckwer, W. D. Journal of Biotechnology 1991, 20 (1), 17–27, DOI:10.1016/01681656(91)90032-Q.
3. Clokie, M. R. J.; Kropinski, A. M. Bacteriophages. Volume 1: Isolation, Characterization,
and Interactions; Methods in molecular biology 501; Humana [u.a.]: Totowa, NY, 2009.
4. Warner, C.; Barker, N.; Lee, S.-W.; Perkins, E. Bioprocess Biosyst Eng 2014, 1-6,
DOI:10.1007/s00449-014-1184-7.
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