Team:Marburg:Project:Notebook:Methods

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Notebook: Methods

Competent Cells

1. Peparation for preculture

  • 50 µL Aliquod in 2x5ml LB each
  • incubation Overnight at 37°C   

2. Preparation for main culture

  • 5ml of preculture in 250ml LB
  • incubation at 37°C until 0,5 OD (2h) →10min on ice

3. Making main culture competent

  • Pellets: 4x50ml in 4 Falcons
  • centrifugation at °C, 3500rpm, 15min
  • resuspending pellets in 4ml HTP* buffer for each Falcon (16ml total)
  • transfer new suspensions in 1 falcon → 10min on ice
  • centrifugation at 4°C, 3500rpm, 15min
  • resuspend pellet in 3ml HTP* buffer
  • add 225 µL DMSO
  • 50 µ aliquods → freeze in N2 (l)
  • store at -80°C
Transformation and Cultivation

1. Transformation

  • transfer 1 µL plasmid into aliquots for 10min on ice
  • 45sec. Heat shock at 42°C
  • 10 min on ice

2. Cultivation of transformed cells

  • add 700 µL LB to transformed cells                                                                                                                                                                     
  • incubation for 1,5h at 37°C
  • centrifugation for 3min at 13000rpm
  • get off 600 µL per 1,5ml tube
  • resuspend pellet in left 100µL LB
  • plating on agar plate
  • incubation overnight
Expression test: Induction + SDS-Gel

1. Picking colonies

  • inoculate 20 ml LB medium (sterile) containing 20µL antibiotics**  and incubate until an OD of 0,6
  • 45sec. Heat shock at 42°C
  • 10 min on ice

2. Induction with IPTG/ Lactose

  • before induction:  0,7/ OD = sample  volume
  • pellet + 80 µL water were resuspended in 20µL loading buffer (preinduction sample)
  • induction with 100 µL IPTG/ lactose at an OD of 0,6
  • incubation for 1,5h    - 4 h max
  • after induction:  200 µL sample   
  • pellet  +80 µL water + 20 µL loading buffer resuspended (induction sample)
  • big volume gap because of  decreased growth of bacteria  
    after induction

3.Expression test with induced culture

  • transfer in 2x50ml falcons
  • centrifugation at 4°C, 3500rpm, 15min
  • washing pellets in 10ml buffer A
  • cracking cells with micro fluidizer
  • thermo centrifugation for 20 min – 4°C
  • 200 µL Ni-NTA beats + supernatant
  • 5min 4000 rpm → pellet
  • resuspended pellet in 500L Buffer A (low imidazole lv)
  • centrifugation 1 min – 4000rpm
  • pellet resuspended in buffer A
  • centrifugation 1 min – 4000rpm
  • pellet resuspended in 200 L Buffer B (elution)
  • centrifugation 1min – 13000
  • 80µL supernatant (incl. protein)+ 20L loading buffer

4. Expression test with induced culture → SDS-Gel

  • ladder – 5 µL
  • PI– preinduction probe - 10µL
  • I    - induction probe -10 µL
  • E    -elution probe -10 µL
Making Competent Bacillus

B. subtilis (PY79) is naturally competent (Albano et al., 1987). In order to increase the transformation rate, Bacillus can be grown in special media to enhance the competence. To achieve this, 20ml SPC-medium was inoculated with half a well grown LB-plate and incubated at 37°C until the cells reached the stationary phase. (Optical density OD550 nm does not change within a a timespan of 30 min) The cells were transfered into 100ml SPII-medium and incubated for 90min at 37°C. Afterwards, the cells were centrifuged (500 rpm, 15min, Rotor: BS4402/A Heraeus). The pellet was resuspended in 1ml glycerine (50% (v/v)). Samples were aliquoted and stored at -80°C.

For the transformation with plasmid-DNA 100µl cells and 5-7µl DNA (chromosomal DNA: 0,1-1µl DNA) were mixed andd incubated at 37°C for 30min. Afterwards, LB-plates (with antibiotics for selection) were inoculated with the culture and incubated at 30°C until colonies formed.

SPC-Medium:

T-Base (10x) 2 ml
D-Glucose Monohydrate 0,5% (w/v)
MgSO4 0,018% (w/v)
Caseinhydrolysate 0,025% (w/v)
Yeast-extract 0,2% (w/v)
ddH2O ad 20 ml

SPII-Medium

T-Base (10x)   10 ml
D-Glucose Monohydrat 50% (w/v)* 1 ml
MgSO4 1,2% (w/v) 7 ml
Caseinhydrolysate 1% (w/v) 1 ml
Yeast-extract 10% (w/v) 1 ml
CaCl2 0,1 M 500 μl
ddH2O ad   100 ml

10x T-Base:

(NH4)2SO4 150 mM
K2HPO4*3 H2O 1 M
KH2PO4 440 mM
Tri- Natriumcitrat di- Hydrat 39 mM

ddH2O ad 1000 ml

*Glucose was substituted with D-fructose in case one of the bacteria strains has a gene inserted under the xylose-promoter, since glucose does inhibit the uptake of xylose

Miniprep (Plasmid DNA Miniprep Kit II, Omega)
  • centrifuge at 10.000g for 1min
  • add 250µl Resuspension Buffer P1 per tube (incl. RNase), vortex
  • transfer suspension into new 1,5 ml tube
  • add 250µl Lysis Buffer P2 per tube, invert until lysate is clear (2-3min)
  • add 350µl Neutralisation Buffer N3 per tube
  • invert immediately (precipitate forms)
  • centrifuge 10min at 13.000 rpm
  • transfer supernatant onto column
  • centrifuge for 1min
  • add 500µl Washing Buffer PE (HBC Buffer) per tube
  • centrifugation for 1min throw filrate away
  • add 700µl DNA Wash Buffer per tube
  • centrifugation for 1min throw filrate away
  • add 30µl milipore per tube – let column sit for 2min
  • centrifuge for 2min
Gel Extraction (QIAquick Gel Extraction Kit)
  • band is cut out of the gel under UV light and transferred into a 1,5ml tube
  • weigh the gel with the tube
  • add QG (100mg gel → 100µl)
  • incubate for approx. 10min at 50-60°C, vortex to dissolve the gel
  • put spin colum into 2mlcollection tube
  • add dissolved gel onto column → centrifuge for 1min
  • discard flow-through
  • wash with 750µl Buffer PE → centrifuge for 1min
  • discard flow-through
  • place column in 1,5ml tube
  • elute with 50µl milipore water: incubate 5min at 37°C → centrifuge for 1min
pMAD-Transformation for Bacillus

1. Preculture for competent Bacillus subtilis

Aim:Preparation for mainculture the next day

5 mL of LB were inoculated with Bacillus WT3610 from an LB plate and incubated at 37°C overnight.

2. Transformation of competent Bacillus subtilis

Aim: transformation of plasmid into Bacillus subtilis WT3610

100 µL of overnight culture were added to 10 mL of MNGE-Medium and incubated till an OD of 1,1-1,3 at 37°C which could take 4-5h.4

After reaching OD of 1,1-1,3 400 µL of the culture were transformed with 1,5 µg plasmid. After 1h incubation at 37°C 100 µL Expression mix were added and incubated for 1h as well.

In the end the 500 µL attempt was plated out on MLS-X-Gal plates and incubated at 30 °C overnight until colonies could be seen.

3. Overnight culture of blue clones

Aim: transformation of plasmid into Bacillus subtilis WT3610

Colonies were grown on the plates with transformed plasmid. The blue/ white screening showed positive transformed blue clones.  3 clones of different morphology per plate were picked and used for inoculation of LB-MLS (4 mL LB, 4 µL Lincomycin, 4 µL Erythromycin). Incubation was carried out overnight at 30°C with the cultures.

4. First temperature shift

Aim: integration of  pMAD-Insert into Bacillus chromosome via flanks

The  overnight cultures were used to inoculate 10 mL LB MLS until  the culture obtained an OD of 0,1. The cultures were incubated at 30°C for 2h.

Then the temperature was shifted to 42°C for 6h.

After the heat shock dilutions from 10-4 to 10-6 of each culture were plated out on MLS-X-Gal so that plates could be incubated overnight at 42°C.

5. Second temperature shift

Aim: flip out of the pMAD backbone

One blue colony per diluted clone was used to inoculate 4 mL LB. The cultures were incubated at 30°C for 6h and afterwards for 3h at 42°C.

Dilutions from 10-4­ to 10-6 were plated out on  X-Gal plates WITHOUT MLS selection. The positive clones should not contain the resistance inside the backbone as well as the galactosidase. The plates were incubated at 42°C overnight.

6.1 selection of positive clones

Aim: checking the correct flip out of the pMAD backbone

From the dilution plates was one WHITE clone picked and transferred on a Master X-Gal Plate as well as on a MLS plate so that clones were proven for the right integration of the insert although flipping out the pMAD backbone.

The plates were incubated at 42°C overnight.

6.2 selection of positive clones

Aim: checking the correct flip out of the pMAD backbone

The white colonies grew on the X-Gal Master plates but not on MLS plates so the transformation seemed to be successful. The Backbone with the MLS resistance flipped out of the genome.

7. cPCR with checked clones

Aim: checking integration of constructs Hag-Spe and Hag-Spe-DARPin into B. s. genome

No clone was growing on MLS plates so the picked clones seemed to be positive. In order to check the correct integration of the domain constructs a cPCR with the picked clones on the X-Gal plates was done. The picked B.s. clones were cooked in 10 µL PBS for 5 min at 95°C.

Making Competent Bacillus with the High Salt/Low Salt Methode

modified after Harwood and Cutting 1990

LB-plates were inoculated with Bacillus subtilies and incubated at 37°C overnight.

3ml HS- (high-salt-) medium were inoculated with a single colony and incubated at 37°C overnight (rolling).

20ml preheated LS- (low salt-) medium was inoculated with 1ml HS-culture and incubated at 30°C for 3h at 100rpm in a water bath.

10 x S-Base

(NH4)2SO4 2g
K2HPO4 14g
KH2PO4 6g
Natriumcitrat* 2 H2O 1g
aqua dest. ad 100ml
After being autoclaved 0,1ml 1M MgSO4 (sterile filtered) were added  

HS-Medium

aqua dest. 74,5 ml
10 x S-Base 10 ml
50% Glucose 1 ml
Tryptophan 5 mg/ml 1 ml
Phenylalanin 3 mg/ml 1,5 ml
Casein-Hydrolysat 2 % 1 ml
Yeast-extract 10 % 1 ml
Arginin 8 % + Histidin 0,4 % 10 ml
Solutions were sterile filtered and mixed under sterile conditions  

LS-Medium

aqua dest. 17,2 ml
10 x S-Base 2 ml
50% Glucose 0,2 ml
Tryptophane 5 mg/ml 20 µl
Phenylalanine 3 mg/ml 30 µl
Casein-Hydrolysat 2 % 0,1 ml
Yeast-extract 10 % 0,2 ml
Spermin 50 mM 0,2 ml
MgCl2 1M 50 µl
Solutions were prepared directly before use and mixed under sterile conditions  

Transformation

Prepare DNA-Solution (1-5µg plasmid DNA or 10-20µg chromosomal DNA) in eppendorf cup.
Add 1ml LS-culture, incubate the transformation samples at 37°C for 2h an a thermomixer/roller.
Centrifuge the transformation samples fpr 20sec, discard some of the supernatant and resuspend the cells.
Incubate selective plates.

(Harwood, C.R., and Cutting, S.M. (1990) Molecular biological methods for Bacillus. John Wiley and Sons Ltd., Chichester, England.)

Transformation of Bacillus subtilis with Spizizens minimal medium
  • 10 ml LB inoculated with single colony as preculture over night at 37°C
  • Inoculate 10 ml Spizizens minimal medium with the preculture to an OD600 of 0.1
  • Incubation at 37°C until OD600 1 more or less
  • Addition of 5 µg plasmid DNA/5-10 µg of chromosomal DNA to 1 ml of culture (in a test tube)
  • Incubation at 37°C for 2 h
  • Plate out 200 µl and 500 µl on different plates containing selecting antibiotics and incubate the plates at 37°C overnight
Purification Ni-NTA column

The following steps were performed for the Ni-NTA Column:

  • equilibration with Buffer A 10 min
  • taking 40 µL supernatant  (load)+ 10 µL SDS-Buffer - L-Probe
  • 50 mL load on column
  • taking 40 µL of flow through + 10 µL SDS-buffer - FT-Probe
  • first washing with 25ml Buffer A (half the load)
  • taking 40 µL of washing flow through + 10 µL SDS-Buffer - W-Probe
  • equilibrate with Buffer B (output pipe off the column into glas, input into B)
  • hanging pipe on column, 20 ml Elution - E
  • taking 40 µL of Elution 1-6 + 10 µL SDS-Buffer E- probe
  • SDS-PAGE analysis

Regeneration of column

  • 10min water
  • 10 min EDTA
  • 10min water
  • 10min NiSO4
  • 10min water
Splitting of Caco-2-cells

Caco-2-cells require DMEM (Dulbecco’s Modified Eagle Medium) with 20% FCS and L-Glutamine.

A centrifuge was set on 4°C.

The medium of the culture flask was discarded from the side opposite to the adherent cells and washed with 1X PBS from the opposite site as well.

The PBS was aspirated with a glass pipette until the supernatant was clear. After adding 1-2 mL 1x trypsin from the opposite of the adherent cells the flask was incubated for 5-10 min at 37°C depending on how fast the cells come of from the ground. Under the microscope the free floating cells were checked.

The cells were transferred into a 15 mL falcon and rinsed with 10 mL DMEM to collect all remaining cells in the flask. The 15 mL falcon was filled with DMEM and the culture spinned down at 1500 rpm  for 5 min at 4°C.

The supernatant was discarded and resuspended in 2 mL DMEM. The cells were splitted 1:3 which means that 666 µL were taken from the suspension and transferred into a new culture flask. After adding 20-25 mL DMEM the flask was incubated at 37°C and checked on the 3rd / 4th day under the microscope.

Purification of PCR Products (QIAquick Gel Extraction Kit) short protocol

short protocol without gel extraction

  • add 3 volumes or QG Buffer to tubes and 1 volume isopropanol
  • place QIAquick spin column in 2ml collection tube
  • centrifuge for 1min →discard flow-through
  • add 750µl Buffer PE
  • centrifuge for 1min →discard flow-through
  • place column in 1,5 ml tube
  • eluate with 50µl milipore water
  • incubate for 5min at 37°C, centrifuge for 1min
Annealing with PNK Inactivation
Step Temperature °C Time
1 37 1h
2 65 20 min
3 95 5 min
4 95-4 1 min/1°C
5 4 Infinite
Medium
LB-medium Concentration Final
Ampicilin Ampicilin (1000x) 100µl:1000ml
Final: 50µg/ml
Canamycin Canamycin (1000x) 100µl:100ml Final: 50µg/ml
Chloramphenicol Chloramphenicol (1000x) 100µl:100ml
Buffer
Buffer Ingredients
-2.0 Buffer 2.0 (10x) + BSA (10mg/ml -10) → BSA end concentration 100µg/ml (0,1µg/µl)
HTP (for 500ml)
10mM Hepes                     1,19g
15mM CaCl2 x2H20 1,1g
250mM KCl 9,32g
55mM MnCl2 x 4 H2O 5,44g
Buffer A 20mM HEPES
250mM NaCl
20mM KCl
20 mM MgCl
40 Imidazole
pH 8,0
Buffer B 20mM HEPES
250mM NaCl
20mM KCl
20 mM MgCl
500 mM Imidazole
pH 8,0
GeFi Buffer 2mM HEPES
20mM NaCl
2mM KCl
2 mM MgCl
pH 7,5
Inclusion Body Wash Buffer 100 mM NaCl
50 mM Tris
pH 8.0
0.5% Triton X-100

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