Difference between revisions of "Team:Freiburg/Experiments"

I) Cloning

1.Culture conditions

The E. coli strains were maintained in lysogeny broth medium. Liquid cultures were incubated at 37 °C and 250 rpm. Solid cultures were incubated on 1.5 % Agar LB plates. Selection of transformed E. coli was performed by supplementation of the LB medium with the antibiotics ampicillin at a final concentration of 100 µg/mL and chloramphenicol at 25 µg/mL.

2.Preparation of chemically competent E. coli

Competent E. coli were prepared using the Zymo Research Mix & Go E. coli Transformation Kit according to the protocol of the manufacturer. The E. coli were inoculated and incubated in 50 mL of ZymoBroth™ medium until an OD600 of 0.6 was reached. The cells were harvested by centrifugation and washed with 1x Wash Buffer. After centrifugation the supernatant was removed and the cells were resuspended in pre-cooled 1x Competent Buffer. The competent E. coli were aliquoted into sterile tubes and stored at -80 °C until further use. Using the chemically competent E. coli prepared by the Mix & Go Transformation Kit no heat shock or electroporation was necessary.

3.Transformation of chemically competent E. coli

The competent E. coli were transformed by addition of 2 – 5 µL of a reaction mix containing the appropriate plasmids. E. coli transformed with plasmids harboring an ampicillin resistance were directly spread on agar plates supplemented with ampicilin. For antibiotic resistances the transformed cells were incubated in 300 µL of LB medium at 37 °C and 250 rpm prior to spreading on a LB agar plate. The spread cells were incubated over night at 37 °C. Subsequently, 5 colonies per plate were picked by a pipet tip, inoculated in 5 mL of LB medium supplemented the appropriate antibiotics and incubated over night at 37 °C and 250 rpm.

4.Bacterial glycerol stocks

For storage of bacterial strains 500 µL of the overnight inoculation were added to 500 µL of 30 % glycerol and gently mixed. The stocks were stored at – 80 °C for later use.

5.Plasmid preparation

The preparation of plasmids from transformed E. coli was performed using the kits from Qiagen, PeqLab or Zymo Research according to the protocols of the manufacturers. Prior to the preparation the transformed E. coli were incubated in 5 mL of LB medium supplemented with the appropriate amount of antibiotics. The cells were harvested by centrifugation and lysed at alkaline conditions. The lysate containing the plasmids was loaded on spin columns and eluted in 30 µL of nuclease free water. The concentration of the DNA was determined by a NanoDrop spectrophotometer.

6.Restriction digestion of plasmids

   a) Digestion of plasmids for cloning

To release inserts from their backbone for further applications 2µg of plasmid DNA was treated with 10 units of the appropriate enzyme in 1X buffer at a total reaction volume of 50 µL and incubated for 1 hour at the appropriate temperature. If two restriction enzymes were used simultaneously the right conditions were determined by the “NEB Double Digest Finder” online tool. The reaction was stopped by addition of 10x orange G loading dye and analyzed by agarose gel electrophoresis.

   b) Test digestion

Verification of transformed plasmids was performed by treatment of 500 ng of the prepared DNA with 5 units of appropriate restriction enzymes in its respective 1x reaction buffer and analyzed by agarose gel electrophoresis. The sizes of the observed bands were compared to the sizes of the expected fragments. Samples that exhibited the expected fragment sizes were further analyzed by sequencing

   c) Linearization of plasmids for transformation of B. subtilis

The transformation of integration vectors into B. subtilis and to insert the fusion constructs into the genome 3 µg of the plasmid was linearized by a single cutter enzyme in the appropriate 1x buffer at a total reaction volume of 50 µL. The reaction was stopped by addition of 10x orange G loading dye, analyzed by agarose gel electrophoresis and subsequently extracted and purified from the gel.

7.Agarose gel electrophoresis

Amplified of digested DNA samples were supplemented with the appropriate amount of 10x Orange G loading dye and loaded on a 1% (w/v) agarose TAE gel. Staining of the DNA was performed by the PeqGreen DNA dye at the recommended concentration. The gel was subjected to electrophoresis at 110 V for approximately 30 min or until a sufficient separation of the bands was observable. Visualization of the separated DNA fragments in the gel was performed by an ultraviolet light imager. A 2-log DNA ladder and a 1kb DNA ladder were included as molecular size marker.

8. Gel extraction

Extraction of amplified genes or digested fragments from agarose gels was performed using the QIAquick gel extraction kit from Qiagen according to the protocol of the manufacturer. The bands corresponding to the expected fragment sizes were excised from the gel, lysed with QG buffer at 50 °C and loaded on a DNA spin column. The DNA was eluted with 30 µL of nuclease free water and stored at – 20 °C until further use.

9. Gibson assembly

In order to generate the fusion constructs the coat genes, the anti-GFP nanobody and the glutathione S-transferase were amplified by extension PCR to introduce overlapping sequences of at least 25 bp required for Gibson assembly. The iGEM standard backbone pBS1C3 was linearized by treatment with XbaI and SpeI. The assembly was performed with the linearized backbone, an amplified coat gene, and the anti-GFP nanobody or the glutathione S-transferase at molar ratios of 3:1 (insert : vector). The volume of the fragments was adjusted to 2.5 µL with nuclease free water and added to 7.5 µL of prepared 1.33X Gibson master mix. The reaction was incubated for 60 min at 50 °C and 2 µL were used to transform chemically competent E. coli DH5α.

10. Polymerase chain reactions

   a) Colony PCR

For the amplification of the genes from B.subtilis a single colony was dissolved in water and lysed by heat shock at 100 °C for 10 min. 1 µL of the lysate was used as template for the amplification. The PCR was performed using the Q5 High-Fidelity 2x Master Mix from NEB, alongside with the appropriate forward and reverse primer at a final concentration of 0.5 µM at a total volume of 50 µL. The reaction was subjected to thermal cycling, subsequently stopped by addition of 10x Orange G loading dye and analyzed by gel electrophoresis.

   b) Extension PCR

In order to introduce additional elements to the used genes, such as linker regions or epitope tags, the PCR was performed using primers with the appropriate overhangs. Approximately, 10 ng of plasmid DNA was used for amplification in Q5 High-Fidelity 2x Master Mix from NEB, alongside with the forward and reverse primer at a final concentration of 0.5 µM in a total volume of 50 µL. The reaction was subjected to thermal cycling, subsequently stopped by addition of 10x Orange G loading dye and analyzed by gel electrophoresis.

   c) Thermal cycling

The thermal cycling was performed according to the recommendations of the manufacturer. Initial denaturation was carried out at 98 °C for 5 min, followed by 30 cycling steps including denaturation at 98 °C for 15 s, primer annealing at the appropriate temperature for 15 s and elongation at 72 °C for 30s/ kb of the amplicon. The cycling was followed by a final elongation at 72 °C for 5 min and the reaction was stored at 8 °C. The annealing temperatures for the used primers was determined by the NEB TM calculator online tool. The thermal cycling was performed as touchdown-PCR. The initial annealing temperature was increased by 10 °C and after each cycle step reduced by 1 °C until the final annealing temperature was reached. backbone

11. T4-Ligation and subcloning

Ligation of amplified B. subtilis coat genes or digested DNA fragments with fusion constructs was performed using the T4 ligase from NEB. The insert, alongside with 50 ng of the linearized backbone were ligated at a molar ratio of 3 : 1 (vector : insert) using 5 units of T4 ligase in 1x T4 reaction buffer in a total reaction volume of 20 µL. The reaction was incubated at room temperature for 30 minutes and 5 µL of the reaction mixture were transformed into chemically competent E. coli DH5α.

12. Annealed oligo cloning

For the insertion of small elements into a vector, two oligos containing the desired sequence were mixed at a molar ratio of 1 : 1 and incubated for 15 minutes at 100 °C, subsequently the temperature was gradually decreased to room temperature over the course of 3 hours. The annealed oligos were ligated into a plasmid with the appropriate overhangs using the T4 ligase from NEB.

13. Sequencing

Plasmids that were verified by restriction digestion were sent to sequencing by GATC biotech. The concentration of the DNA samples was adjusted to 30 – 50 ng/µL at a total volume 20 µL. The appropriate primer required for the sequencing reaction were sent in an additional microtube at a concentration of 10 µM. The sequencing results were analyzed using the Geneious software.

II) Bacillus subtilis

1. Cultivation and germination of Spores

The Bacillus subtilis strains were grown and kept on 1,5% LB. The dilutions were grown in the incubator at 37°C with 200 rpm. To germinate spores, the samples were also diluted with LB and incubated over night.

2. Sporulation

The production of spores from Bacillus subtilis takes up to 24 hours. The samples were diluted in LB at 37°C with 200 rpm until they reach their exponential growth phase and then put in difco sporulation medium (DSM) which exerts pressure on them. After 24 hours of incubation most of the cells are sporulated.

3. Competent cells + Transformation

To make Bacillus subtilis competent, the samples were diluted in minimal medium and grown until they reached an OD600 of 1,0-1,3/ml. 1000ng of DNA was added to 400ul of the competent cells, incubated and streaked on selective agar.

III) Expression analysis

IV) Targeting

1. (3-Glycidyloxypropyl)trimethoxysilane (GOPTS) - Coating of microscope slides

Glass slides were washed with EtOH (70%) and ddH2O and activated by oxygen plasma (300s, 50% Power, Plasma Cleaner ZEPTO, Electronic Diener). The activated surface was incubated with GOPTS (50µl, 2h, darkness). The slides were then washed with acetone (99,9%) to remove excess GOPTS.

2. Spotting

A spotting mask (silicone, CultureWell™) was used to evenly create sample spots on slides. The spots were incubated with protein solution (GFP/anti-GFP nanobody) (2µl, 15 min, 4°C, darkness) of different concentrations. Blocking was done with a BSA (4%)- or milk protein (5%)-solution.

3. Spore binding assay

A solution of spores (of different concentrations) was pipetted on a nanobody spot and incubated for 20 min. Excess spore were washed off using PBS(1x) (Phosphate Buffered Saline) (10 min, 10 rpm).

4. Sample preparation

After washing the slides were dried with compressed air. A Mowiol/DABCO (2.5%) mounting solution was used to avoid withering of the proteins (amount depending on size of the cover slip). The samples have been incubated overnight. The coverslips were sealed with nail polish.

5. Fluorescence microscopy

Sample analysis was done by fluorescence microscopy (Nikon C2+ confocal).

V) Delivery