Glycerol stocks – storage of bacterial strains

Material:

• Glycerol

Method:

1. Mix 700 µL of overnight culture with 300 µL glycerol.
2. Store at -80°C

Polymerase chain reaction (PCR)

Material:

• Primer forward & reverse
• Template DNA
• NEB Q5® High-Fidelity 2X Master Mix (dNTPs + Polymerase)
• Distilled water
• PCR-Cycler

Method:

1. All steps have to be performed on ice.
2. 50 µL approach (mix well):

Components	Volume	Concentration
Q5 Master Mix	25 µL	1x
10 µM fw primer	2,5 µL	0,5 µM
10 µM rv primer	2,5 µL	0,5 µM
Template DNA		<1000 ng
Water	remaining volume to 50 µL

PCR-cycler conditions:

Step	Cycles	Temperature	Time
Denaturation	1	98°C	30 sec
Annealing	25-35	98°C	5-10 sec
Elongation		72°C	20-30sec/kb
Final extension	1	72°C	2 min
Hold		4-10°C

Determination of DNA concentration

• DNA concentration is determined by using a Nanodrop ()
• The absorbance at 260 nm is converted to concentration using the Lambert – Beer Equation by the program
• The purity of the sample is confirmed by the 260/280 ratio (contamination with proteins) and the 260/230 ratio (presence of co-purified contaminants)
• For pure DNA the 260/280 ratio should be around 1.8 and the 260/280 ratio should be around 1.8 – 2.2. (ND-1000-v3.4-users-manual, Thermo Scientific)

Mini-Prep (based on Fast-n-Easy Plasmid Mini-Prep Kit Jena Bioscience)

Material:

• Lysis buffer
• Neutralization buffer
• Column-Activation buffer
• Wash buffer
• Elution buffer
• Binding column

Method:

1. Harvest the over-night culture by centrifugation (3000 g for 10 minutes.)
2. Activate the Binding Column by adding 100 µl of Activation buffer and centrifugation at 10000 g for 30 seconds.
3. For cell lysis resuspend the cell pellet in 300 µl Lysis buffer (pipetting or vortexing).
4. Add 300 µl of Neutralization buffer and mix by inverting the tube (4 – 6 times).
5. Centrifuge at 10000 g for 5 minutes.
The colour of the supernatant should be yellow.
6. Transfer the supernatant into the Binding Column and centrifuge at 10000 g for 30 seconds. Discard the flow-through.
7. Add 500 µl Washing buffer to the column and centrifuge at 10000 g for 30 seconds. Discard the flow-through.
8. Place the Binding Column into a clean microtube an add 30 µl Elution buffer.
9. Incubate for 1 minute and centrifuge at 10000 g for 1 minute to elute DNA.
10. The eluted DNA could be used directly or should be stored at -20°C.

Restriction digest

Material:

• Plasmid DNA
• Restriction Enzymes
• Restriction buffer
• H2O
• Ice

Method:

1. All steps must be performed on ice.
2. For a 20 µl double digest approach following amount are needed:



Components	Volume	Concentration
Restriction buffer (10X)	2 µL	1X
Restriction enzyme 1	1 µL
Restriction enzyme 2	1 µL
Plasmid DNA	x µL	1 µg total
H2O	add to 20 µL



3. Digest the approach for 1h at 37 °C.
4. To stop the reaction, incubate the reaction for 20 minutes at 80 °C.

Gel-Extraction (based on Gel DNA Recovery Kit from Zymo Research)

Material:

• Extraction buffer (ADB buffer)
• Washing buffer
• Elution buffer
• Spin column

Method:

1. Cut out the area of the gel containing the DNA fragment of interest.
2. Add ADB buffer (3 volumes to 1 volume gel).
3. Dissolve the gel by incubation at 37 – 55 °C for 5 – 10 minutes.
4. Transfer the solution to a spin column and centrifuge for 30 seconds. Discard the flow-through.
5. Add 200 µl Washing buffer and centrifuge for 30 seconds. Discard the flow-through.
6. Repeat the wash step.
7. Place the column in a clean microtube and add more than 6 µl Elution buffer. Centrifuge for 1 minute to elute DNA.

Enzyme activity assay of the esterases EstCS2 (BBa_K1149002) and LipB

To determine the enzyme activity of EstCS2 and LipB a photometric assay was prepared. Two wildtypes were compared with the enzyme activity of the esterases. The results were obtained from biological triplicates.

Principle

Material:

• Agar plates
• Spectrometer
• Photometer
• Centrifuge
• 96 well plates

Chemicals:

• LB media
• PBS buffer
• Arabinose stock solution [500 mM]
• IPTG stock solution [1 M]
• p-nitrophenyl butyrate [50 mM] diluted in acetonitril

Method:

Preparation of the cell lysate

1. Pick a colony from the agar plates
2. Grow the colony in 5 mL LB media (+antibiotic) over night
3. Dilute the overnight culture 1:100 in 15 mL LB media (+antibiotic)
4. Grow the cells until the OD600 reached 0,2
5. Induce gene expression with IPTG/arabinose with different concentrations (0 mM, 1 mM, 2 mM, 3 mM) over night
6. Grow the cell culture until the OD600 reached 6
7. Harvest the cells by centrifugation (10 min, 3000 rpm)
8. Resuspend the cell pellet in 10 mL PBS buffer

Enzyme activity assay: Continous spectrometric rate determination

1. 100 µL of the supernatant (1:10 dilution) or resuspended cell pellet and 10 µL p-nitrophenyl butyrate with different concentrations [2.5; 5; 10; 15; 20 mM] are mixed in a 96 well plate
2. Photometric enzyme activity test with the following conditions: wavelength: 405 nm, temperature: 37 °C time: measurement every 45 seconds for 30 min 96 well plate is shaked (200 rpm)
3. Calculation of the enzyme activity out of the absorption rates. 1 unit (U) was defined as the amount of enzyme that catalyzes the reaction of 1 nanomol of substrate per minute.

Determination of protein concentration - BCA Assay (Pierce™ BCA Protein Assay Kit – Thermo Scientific)

Material:

• BCA reagent A and B (provided in Assay Kit)
• BSA stock solution (provided in Assay Kit)
• 96-Well microplates
• Microplate reader
• Microplate incubator/shaker

Method:

1. Preparation of serial dilution of BSA-standard:

Vial	Volume of Diluent (µL)	Volume and Source of BSA (µL)	Final BSA Concentration (µg/mL)
A	0	300 of Stock	2000
B	125	375 of Stock	1500
C	325	325 of Stock	1000
D	175	175 of vial B dilution	750
E	325	325 of vial C dilution	500
F	325	325 of vial E dilution	250
G	325	325 of vial F dilution	125
H	400	100 of vial G dilution	25
I	400	0	0=Blank

Table: User Guide, Pierce™ BCA Protein Assay Kit – Thermo Scientific


2. Preparation of Assay Working Reagent: mix 50 parts of Reagent A with 1 part of Reagent B.
3. If necessary dilute your samples to fit into the working range of 20-2000µg/mL.
4. Pipette 25 µL of each standard and sample into a microplate and add 200 µL of Working Reagent.
5. Mix for 30 seconds on plate shaker and incubate at 37°C for another 30 minutes.
6. After cooling down to room temperature, measure the absorbance at 562 nm.

Semi-quantitative hair degradation assay for keratinases

To prove any enzyme activity a semi-quantitative hair degradation assay was performed. First, cultures of E. coli containing kerA, kerUS, kerP plasmid and one wild-type E. coli were grown at 37°C in sterile LB broth. Chloramphenicol (final concentration 35 µg/mL) was added to the cultures containing kerA and kerUS. Kanamycin (final concentration 50 µg/mL) was added to the cultures containing the kerP. After incubation OD600 was measured before inducing cultures containing kerA and kerUS with IPTG with a final concentration of 1mM. Human hair was reduced to smaller pieces and then dried for 1 hour at 65°C. Afterwards the hair was distributed in 0.05 g aliquots and the full amount of each culture was added. The cultures + hair were incubated 120 hours at 37°C with slightly shaking.

Skim milk plate assay for keratinases

This assay was performed to show qualitative enzyme activity. The different keratinases (kerA, kerUS and kerP) should degrade the casein in the milk, seen as clear zones around the cultures itself or the supernatant of the cells.

Preparation of skim milk plates:

First 100 mL LB/agar and 5 g skim milk powder in 125 mL distilled water was prepared. Both solutions were autoclaved and mixed after cooling down. For the plates used for kerA and kerUS cultures chloramphenicol (35 µg/mL) was added – in case of kerP kanamycin (50 µg/mL) was used to prepare plates. The plates were poured and stored at 4°C.

Assay:

• kerA/kerUS: Before spreading both keratinases on the plates, cells were induced with 1mM IPTG o/n at 37°C. After incubation 10 µL of the induced cell cultures and supernatant, cell culture without IPTG induction and a wild-type E. coli control was spread on skim milk plates containing chloramphenicol. The plates were incubated at room temperature for four days.
  
• kerP: 10 µL of cell culture, supernatant and a wild-type E. coli control was spread on skim milk plates containing kanamycin. The plates were incubated at room temperature for four days.