Materials and Methods

Wetlab protocols

Bacteria strains

Five bacterial strains: Escherichia coli DH5ɑ, Escherichia coli BL21, Bacillus subtilis DSM 10, Bacillus subtilis 168 and Acinetobacter baylyi ADP1 were cultured. All five species can be grown in autoclaved LB medium. In liquid culture at 180 rpm shaking and on agar plates, both at 37 °C. Using an autoclaved R2A medium at 28 °C 180 rpm shaking results in optimal growing conditions for A. baylyi.

LB medium ingredients

	for 500 mL
Bacto Tryptone/Peptone	5 g
Bacto yeast	2.5 g
NaCl	5 g
Millipore H₂O	500 mL

For LB medium with antibiotics, 10 μL ampicillin (53 mg/ml), 10 μL carbenicillin (53 mg/ml) or 10 μL kanamycin (30-50 mg/ml) were added to 5 mL medium. Store at room temperature.

LB agar plates ingredients

	for 500 mL
Bacto Tryptone/Peptone	5 g
Bacto yeast	2.5 g
NaCl	5 g
Agarose	5 g (1%)
Millipore H₂O	500 mL

For LB agar with antibiotics add 1 mL ampicillin (53 mg/ml), 1 mL carbenicillin (53 mg/ml) or 1 mL kanamycin (30-50 mg/ml) to the autoclaved medium before filling the plates. Store plates at 4 °C.

R-2A medium ingredients

	for 500 mL
R-2A broth	1.56 g
Millipore H₂O	500 mL

R-2A broth from the brand HiMedia was used. Store at room temperature. For R-2A medium with antibiotics, 10 μL ampicillin (53 mg/ml), 10 μL carbenicillin (53 mg/ml) or 10 μL kanamycin (30-50 mg/ml) were added to 5 mL medium.

R-2A agar plates ingredients

	for 500 mL
R-2A broth	1.56 g
Agarose	5 g (1%)
Millipore H₂O	500 mL

R-2A broth from the brand HiMedia was used.

For R-2A agar with antibiotics add 1 mL ampicillin (53 mg/mL), 1 mL carbenicillin (53 mg/mL) or 1 mL kanamycin (30-50 mg/ml) to the autoclaved medium before filling the plates. Store plates at 4 °C.

Overnight culture

Bacteria for overnight cultures were either picked from plates using a pipette tip or taken from glycerol stocks. Liquid medium and temperature were adjusted to the strains (A. baylyi: R-2A, 28 °C; A. baylyi, B. subtilis, E. coli: LB, 37 °C). The volume of liquid medium used was 3 mL in a reaction tube with an air permeable lid or 50 mL in an Erlenmeyer flask with aluminium foil lid.

Streaking

Streaking was performed with two methods: For plating a drop of culture a Drigalski spatula was used to spread the solution evenly on the agar plate. This method yielded often, but not always single colonies, therefore dilution plating was used, when single colonies were needed. With a glass loop the sample was smeared on the plate in a zig-zag pattern on one third of an agar plate. A clean glass loop was then smeared through the already applied bacteria and smeared on the next free third of the plate. This was repeated for the last third of the plate.

Plate reader assays

For absorbance assays a NanoQuant infinite M200Pro plate reader from Tecan with i-control 1.11 software was used.

For fluorescence assays an infinite F200Pro plate reader from Tecan with i-control 1.11 software was used.

96 well plates; transparent and black,with U or flat bottom, were used depending on the assay. The maximum fill volume per well was 200 μl.

Glycerol stocks

500 μL overnight culture was added to 500 μL 50% glycerol solution in a 1 mL cryo tube. The cryo tubes were cooled on ice before being stored at -80 °C.

Plasmid preparation

The kit used for plasmid preparation is the QIAGEN® Plasmid Plus Midi Kit. It was used mostly according to the manufacturer's instructions with 50 mL overnight culture. For the last step (eluting plasmid DNA from the column) 100 μL instead of the 200 μL as written in the manual were used.

Generating competent E. coli bacteria

1 mL overnight culture of E. coli was added to 50 mL fresh LB medium and grown to an OD600 of 0.6. At this OD the bacteria are in the exponential growth phase. The culture was centrifuged at 10.000 rpm for 5 minutes at room temperature and the supernatant discarded. The pellet was resuspended in 5 mL of TSB buffer and incubated for 10 minutes on ice. 100 μL cell suspension was added to 1.5 mL reaction tubes that were pre-cooled on dry ice before immediately transferring to storage at -80 °C.

TSB buffer (1x) ingredients

	for 50 mL
2x LB medium	25 mL
DMSO	2.5 mL
MgCl₂	0.5 mL, 1 M
MgSO₄	0.5 mL, 1 M
PEG4000	5 g
H₂O	fill up to 50 mL

Filter with 0.2 μm for sterilization of buffer.

E. coli heat shock transformation

Aliquots of competent E. coli and the plasmid were defrosted on ice. For the negative control 20 μL (5x) KCM buffer was added to 80 μL H₂O. For transformation 50 ng plasmid were added to 20 μL (5x) KCM and 80 μL H₂O. 100 μL competent E. coli were added to each buffer and the solution was incubated on ice for 40 minutes. After that, the solutions were incubated on a 42 °C heating block for 45 seconds before transferring them back to the ice for 5 minutes. 1 mL LB medium was added to each reaction tube and they were transferred to a 37 °C, 300 rpm heating block for 1 hour. After that, the solution was centrifuged at 3.000 rpm for 2 minutes and the supernatant discarded. The bacterial pellet was resuspended in 110 μL LB medium and diluted 1:10 and 1:50. All dilutions were plated on LB agar plates with antibiotics and incubated at 37 °C overnight.

KCM buffer (5x) ingredients

	concentrations
KCl	500 mM
CaCl2	150 mM
MgCl2	250 mM

Agarose gel electrophoresis ingredients

	for one gel
Agarose	0.3 g for 1%, 0.6 g for 2%
TAE (1x)	30 mL
heat until dissolve in the microwave
Ethidium Bromide	0.6 μL

DNA ladders from New England BioLabs (NEB) (1 kb) and Jena Bioscience (1 kb, 50 bp) were used. The gel loading dye purple (6x) from NEB was used. The loaded gel was run with 100 V for about 25 minutes. The gel assembly is based on the system from Bio-Rad.

Restriction enzymes

Six restriction enzymes were used: EcoR1_HF, Nde1, BamHI, BsaI, PvuI and NcoI. The protocols are shown in the tables below.

EcoR1_HF mix

	for 1 μg DNA
CutSmart buffer 10x	5 μL
EcoR1_HF	1 μL
DNA	1 μg
Nuclease free H₂O	0.6 μL

EcoR1_HF and CutSmart buffer are from New England BioLabs (NEB).

Temperature	Time
37 °C	5-15 min
65 °C (inactivation)	20 min

NdeI + BamHI mix

	for 1 μg DNA
RE 10x Buffer D	2 μL
BSA (10 μg/μL)	0.2 μL
DNA	1 μg
BamHI	0.5 μL
NdeI	0.5 μL
Nuclease free H₂O	fill up to 20 μL

NdeI,BamHI and Buffer D were bought from Promega.

Temperature	Time
37 °C	2 h
65 °C (inactivation)	15 min

BsaI mix

	for 1 μg DNA
CutSmart buffer 10x	5 μL
Bsal (10 μg/μL)	1 μL
DNA	1 μg
Nuclease free H₂O	fill up to 50 μL

BsaI and CutSmart buffer were from New England BioLabs (NEB).

Temperature	Time
37 °C	5-15 min
80 °C (inactivation)	20 min

PvuI + NcoI mix

	for 1 μg DNA
3.1 buffer 10x	2 μL
Ncol	1 μL
Pvul	1 μL
DNA	1 μg
Nuclease free H₂O	fill up to 50 μL

Restriction enzymes and 3.1 buffer were from New England BioLabs (NEB).

Temperature	Time
37 °C	5-15 min
80 °C (inactivation)	20 min

Polymerase chain reaction (PCR)

Ruby Hot start Master from Jena Bioscience was used according to manufacturer's instructions, adjusted to template length and primer annealing temperature.

	for 50 μL
Ruby Hot start Master 2x	25 μL
Primer Mix (forw & rev) (10 μM)	0,5 μl of each primer (total 1 μl)
Template DNA	<20 ng (1-15 ng used)
PCR-grade H₂O	fill up to 50 μL

Step	Temperature	Time
Initial Denaturation	95 °C	1 min	1x
Denaturation	95 °C	15 sec	25x
Annealing	depending on primer	15 sec	25x
Elongation	72 °C	1 min/kb	25x
Final Extension	72 °C	5 min
Hold	4 °C	∞

For colony PCR the initial denaturation time was increased to 7 minutes.

Overlap extension PCR (OEPCR)

For the OEPCR we used the pfu polymerase from promega according to manufacturer's instructions, adjusted to template length and primer annealing temperature.

	for 50 μL
pfu Polymerase (2-3 u/μl)	0.5 μL
Template DNA Vector	10 ng
Template DNA Insert	10 ng
dNTP mix (10 mM)	1 μL
pfu Polymerase Buffer 10x	5 μL
PCR-grade H₂O	fill up to 50 μL

Step	Temperature	Time
Initial Denaturation	95 °C	2 min	1x
Denaturation	95 °C	45 sec	25x
Annealing	depending on primer	30 sec	25x
Elongation	73 °C	2 min/kb	25x
Final Extension	73 °C	5 min
Hold	4 °C	∞

PCR cleanup

For clean up of the PCR products Monarch PCR & DNA Cleanup Kit from New England Biolabs was used according to manufacturer's instructions.

Gel extraction

For extraction of DNA from agarose gels two kits were used: Wizard® SV Gel and PCR Clean-Up System from Promega and Agarose Gel Extraction Kit from Jena Bioscience.

Ligation

For ligation T4 buffer and T4 DNA ligase from New England BioLabs (NEB) were used.

	for 20 μL
Vector DNA	50 ng
Insert DNA	37.5 ng
T4 Buffer 10x	2 μL
T4 DNA ligase	1 μL
Nuclease free H₂O	fill up to 20 μL

Incubate mixture for 20 minutes at 22 °C before inactivating the ligase at 65 °C for 10 minutes.

X-Gal Assay/ blue-white screening

0.0025 g X-Gal were dissolved in 125 μL DMSO to get a 20 mg/mL solution. LB agar plates were prepped with 40 μL 20 mg/ml X-Gal and 300 μL 0.1 mM IPTG. After that E. coli with pUC19 plasmid were spreaded and incubated at 37 °C overnight.

AvPAL assay in vitro

The PAL assay in vitro was performed by first rupturing in a french press E. coli expressing PAL and collecting the supernatant. The supernatant was pipetted on a 96 well plate together with different amounts of phenylalanine to get various concentrations between 0 to 1 mM. Over a 20 hour period the absorption at 300 nm was measured every 30 minutes in the Tecan listed above under “Plate reader assay”.

3,5-dinitrosalicylic acid (DNS) method

This assay is used to indicate the presence of reducing sugars that are produced when β-agarase degrades agarose. First a standard curve was created with galactose in concentrations between 0 to 2.5 mg/mL. The DNS-reagent was prepared by dissolving 45 g of sodium potassium tartrate in 75 mL H₂O and dissolving 1.5 g of 3,5-dinitrosalicylic acid in 30 mL of 2 M NaOH. After that both solutions were combined. To 3 mL of sugar solution in a test tube 1 mL of DNS-reagent was added and the mixture in the test tubes submerged in boiling water for 5 minutes. The solutions were cooled down to room temperature and absorbance was measured at 540 nm. For the agarase assay in vivo and in vitro 4 mL 4% agarose gel was added to the bottom of three autoclaved 25 mL erlenmeyer flasks. To one flask 3 mL LB medium was added as a negative control, to another one 3 mL supernatant of a centrifuged (10.000 rpm, 5 min) overnight culture of E. coli with ß-agarase and to the third flask E. coli with or without ß-agarase in 3 mL LB medium was added. The three erlenmeyer flasks were incubated at 37 °C, 40 rpm overnight. After incubation all three supernatants were centrifuged at 10000 rpm for 5 minutes. To 1.5 mL of supernatant 0.5 mL of DNS-reagent was added and the mixture boiled at 100 °C for 5 minutes. After the solution has cooled to room temperature the OD540 was measured.

Co-culture assay

To measure the growth of different bacterial species, strains expressing GFP, EGFP, mRFP1 or mCherry were grown in overnight cultures. In the beginning of the assay, the bacterial strains were pipetted on a black 96-well plate with flat bottom in different ratios: 100:0, 0:100, 50:50, 80:20 and 20:80 and incubated at 37 °C with shaking in the Tecan for fluorescence measurements. Every 30 minutes the emission at 535 nm (EGFP, GFP) and 635 nm (mRFP1, mCherry) was measured.

Selective advantage assay

**Figure 4: Overview of the selective advantage experimental setup.** It is tested whether or not genes that give a bacteria strain the ability to degrade otherwise inaccessible sugars improves their viability and let them overgrow other strains. Created with BioRender.com

Two genes were tested in this assay: agarase and glucosidase. As a comparison mCherry was used.

E. coli expressing the genes to test were cultured together at 37 °C, 180 rpm. Samples were taken at 0 h, 4 h, 6 or 7 h and 24 h, diluted and spread on LB agar plates with X-Gal. The agar plates were incubated at 37 °C overnight and the colony forming unit (CFU) counted. Colonies with agarase have a small dip around them, colonies with mCherry are red and colonies with glucosidase appear white.

Natural transformation assay A. baylyi ADP1

**Figure 5: Natural transformation assay.** *Acinetobacter baylyi* ADP1 is a natural competent bacteria species. It can take up plasmid DNA from its surroundings. Created with BioRender.com

70 μL A. baylyi overnight culture was added to 1 mL fresh LB medium and plasmid DNA added to the solution. For the “log-phase” protocol the culture was incubated for 5 hours and then the plasmid was added for 3 hours. For the second “overnight” protocol the plasmid was added directly to the dilution for 5 hours and then streaked out. For the plasmid DNA two different amounts (0, 25 and 100 ng) were used. These mixtures were grown overnight and plated on LB agar with appropriate antibiotics. This “overnight” experiment setup was used with a co-culture of E. coli, B. subtilis and A. baylyi. The plasmid DNA used was increased to 100 ng and 200 ng.

Lastly natural transformation on LB agar was tested. A. baylyi and B. subtilis (with GFP) were streaked on a LB agar plate in lines. Once streaked out in parallel and the other time in an X-pattern. After one day of incubation at 37 °C, mCherry plasmid was added on top of the A. baylyi lines. After another day of incubation at 37 °C fluorescence was observed with a UV-light table.

Sequencing

We prepared plasmids to be sent to Eurofins for sequencing according to the manufacturer's instructions.

SDS-PAGE

To lyse the bacteria 50 mL overnight culture the culture was centrifuged 3.270 rpm for 15 minutes at 4 °C. Then the pellet was lysed in DPBS 1x and fracked using a french press machine and centrifuged at 20.000 rpm, 4 °C for 2 hours.

SDS-PAGE ingredients

	Separation gel 5 mL/gel	Separation gel 2 mL/gel
H₂O	7 mL	4.4 mL
Tris buffer 1,5 M, pH 8.8	5 mL	/
Tris buffer 0,5 M, pH 6.8	/	2 mL
Acrylamide	8 mL	1.6 mL
SDS 10%	200 μL	80 μL
APS 10%	100 μL	40 μL
TEMED	10 μL	4 μL

SDS-PAGE Buffer (10x) for 1L ingredients

	Amount
Tris	30 g
Glycerine	144 g
SDS	10 g

APS and TEMED were added last, as they start the polymerisation. At first the separation gel was prepared. After 20 minutes isopropanol was applied on top of the separation gel to prohibit drying out. After additional 25 minutes the isopropanol was removed with a Whatman paper and the stacking gel prepared. After the addition of the stacking gel, the comb was attached. The gel assembly is based on the system from Bio-Rad.

40 μl of the samples were mixed with 10 μl of the loading dye (5x) or diluted appropriately. The loading dye contained ß-mercaptoethanol, sodium dodecyl sulfate (SDS), glycerol and bromophenol blue. 10 μl of the sample were loaded into the chambers. The PrecisionPlus Protein™ Dual Color molecular mass marker from Bio-Rad was added with 4 μL. The SDS-PAGE ran 3 hours at 100 V.

Before staining with Coomassie Blue, the western blot transfer was performed.

Western Blot

For the western blot, samples were first run on an SDS-PAGE. The PVDF membranes and filter paper were cut to the right size fitting in the Bio-Rad Trans-Blot® Turbo™ transfer system machine. Then, the packing of the filter paper, the membrane and SDS-PAGEs were prepared in the right order for the semi-dry transfer. First three layers of filter paper, then the SDS-PAGE gel, then the PVDF membrane and finally three more layers of filter paper. Transfer buffer needed to be added and streaked out to every layer except on the membrane. The blotting was performed for 22 minutes at 20 V. After the blotting, the following two washing steps with demineralized (VE) water took five minutes each and the membrane was blocked with bovine serum albumin (BSA) 5% for one hour with shaking. The membrane was then treated with the primary antibody (mouse anti-his antibody) solution at 4 °C overnight. The membrane was washed three times in PBST buffer for five minutes and incubated with the secondary antibody (anti-mouse antibody with reporter) for one hour at room temperature on the shaker. Afterwards the membrane was washed again three times with PBST for five minutes each. The membrane was directly treated with the ECL prime reagent. The western blot membrane luminescence reaction was visualized using a ChemiDoc™ MP Imaging system from Bio-Rad.

Transfer Buffer (10x) ingredients

	Concentration
Tris	0.25 M
Glycerine	1.92 M
set to pH 8.6

PBST- Buffer (10x) ingredients

	for 1 L
Sodium Chloride	80 g
Potassium chloride	2 g
Disodium hydrogen phosphate	14.4 g
Tween	0.01%
Set to pH 8.6

Coomassie Blue staining

SDS-PAGE was stained in Coomassie Blue solution for 1 hours while shaking at room temperature. Then the SDS-PAGE was bleached with 10% acetic acid for minimum 3 times for 1 hour until bands were recognizable.

Ponceau staining

The staining is used to control whether the blotting was successful. The PVDF membrane was stained with the ponceau solution for 1 hour with shaking at room temperature and discolored with distilled water.

Team:Heidelberg/Methods Materials