Difference between revisions of "Team:Goettingen/Experiments"

Revision as of 11:20, 11 October 2016

Experiments

Overview of Experiments

1. Production Strains

In order to ensure that the bacteria we are working with are really the ones we want to work with, a 16S rDNA PCR was performed.

As an antibiotic resistance will be used in our later experiments to select transformants, we checked for any native antibiotic resistance against ampicillin, kanamycin, tetracyclin and chloramphenicol in an LB agar plate growth test. It turned out that kanamycin was the only antibiotic, against which none of our strains showed any resistance. For this reason, we decided to use only vectors with kanamycin resistance for our later projects.

After this, we prepared electro competent cells from our strains. All of our strains were grown on LB medium.

2. BioBricks

Our BioBricks were designed using the software SnapGene. The original Genes for our B₁₂ binding proteins, and the torA signal sequence were codon optimized for E. coli.

The DNA containing the Genes were synthesized and friendly provided by IDT Integrated DNA Technologies.

In case of MutB, the length of the gene exceeded the maximum length of 2000 bp from IDT. For this reason, we designed two DNA parts, with a natural occurring HindIII restriction site at both ends. We intended to fuse these both genes by ligation. However, after having diffuculties to fuse the genes by ligation, we decided to fuse them by fusion PCR. In order to multiply the DNA for or cloning experiments, and to equip our genes with restriction sites, PCR was used.

For our purpose, we were searching for a vector with (a) kanamycin resistance and (b) araBAD operon. We thus decided to use the pBAD202 expression system. This vector, however, is equipped with a thioredoxin fusion protein which increases the rate of expression, but prohibits the physiological function of our protein. For this reason, we designed primers for an autarkic synporter protein. In order to test the dependence of the expression system on the B₁₂ export rate, we also decided to use different other vectors.

For vectors ligation, we used a standart ligase reaction, for the pBAD202 expression, which works without ligation but topoisomerase reaction, we followed the manufacturer's protocol.

3. Transformation

For our transformation experiments, we used electroporation for all our self prepared competent cells. For the heat competent E. coli Top10 cells provided with the pBAD202 expression system, we used the manufacturer's heat shock transformation protocol.

Protocols

Media

LB Medium

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M9 Minimal Medium

Guttman Medium (B₁₂ Assay)

S. blattae Medium Anaerobic

Cultivation and Transformation

Cultivation of Escherichia coli, Shimwellia blattae and Salmonella typhimurium TA 100

Cultivation of Raoultella planticola

Preparation of Electrocompetent Cells

Material to prepare

2x 5 mL LB
250 mL LB in 1 L Erlenmeyer flask with chicane
500 mL sterile Millipore-H₂O at 4 °C
50 mL sterile glycerol (10 % (w/v)) at 4 °C
30 sterile labeled Eppendorf cups at – 20°C or -80 °C
1x sterile GS3 jar at 4 °C
1x Aquatron at 30 °C
1x Sorvall RC6 centrifuge at 4 °C
1x Universal 320R centrifuge at 4 °C
1 L liquid nitrogen

Day 1

Inoculate 2x 5 mL LB (if required with antibiotics) with your strain of interest, either from cryo culture or from agar plate.
Incubate overnight at 37 °C and 150 rpm.

Day 2

Inoculate 250 mL LB (without antibiotics) with 2 % (5 mL) preculture.
Incubate in the Aquatron at 30 °C and 160 rpm until OD₆₀₀ of 0.5-0.8.
Check the culture via microscope for contaminations.

All liquids and containers must be cooled on ice. The major task of cell preparation is the removal of salts. In case of some strains like pLys-strains, the pellets must be resuspended very carefully.

Let the cells (OD₆₀₀ of 0.5-0.8) cool down in ice water for 10-20 min. All further steps are performed under cool conditions.
Decant the cells in sterile GS3 jar and centrifuge at 4 °C and 5000 rpm (4230 g) for 5-10 min.
Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 250 mL sterile 4 °C ddH₂O.
Centrifuge the GS3 jar at 4 °C and 5000 rpm (4230 g)for 5-10 min.
Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 250 mL sterile 4 °C ddH₂O.
Centrifuge the GS3 jar at 4 °C and 5000 rpm (4230 g)for 5-10 min.
Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 10 mL sterile 4 °C glycerol (10 % (w/v)). Afterwards, transfer the culture into a 50 mL Falcon tube.
Centrifuge the Falcon tube at 4 °C and 6200 rpm (4230 g) for 5-10 min.
Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 10 mL sterile 4 °C glycerol (10 % (w/v)).
Centrifuge the Falcon tube at 4 °C and 6200 rpm (4230 g) for 5-10 min.
Remove supernatant (directly next to the centrifuge or the pellet might resolve) and resuspend the pellet in 500 µL sterile 4 °C glycerol (10 % (w/v)).
Aliquot the cells into the Eppendorf cups (40 µL per Eppendorf cup). While aliquoting, filled Eppendorf cups must be frozen directly in liquid nitrogen.
Store the filled Eppendorf cups in a cryobox at -80 °C.

Transformation Electroporation of Electrocompetent Cells

Mix 50-200 ng plasmid DNA with 40 µL of competent cells (always defrost on ice!). Transfer attempt into precooled electroporation cuvette. Incubate on ice for 10 min.
Important: DNA must be salt free! Use either directly in ddH₂O eluted plasmids, or (e.g. directly after ligation) desalt the DNA for 30 min on a Millipore filter (MF Membrane Filters).

Electroporation: 2500 V for 2 mm cuvettes (1250 V for 1 mm cuvettes), 25 µF, 200 Ω, discharging time should be 3-5 msec. Contacts of the electroporation cuvette must be dry. Remove all air bubbles before electroporation.
Immediately add 960 µL liquid LB (or, if available, SOC medium for a higher transformation efficiency) into the cuvette and transfer the content into a 2 ml Eppendorf cup.
Incubate at 37 °C and 150 rpm for 1 h. Fix tube in horizontal position with tape.
Plate 100 µL of 10^-3 to 10^-6 dilutions on plates with selection pressure
Incubate at 37 °C overnight.

Heat Shock Transformation of Heat Competent Cells

PCR and Cloning

Entry 1

ENtry 2

Entry 3

Entry 4

Analytic Tests

Agarose Gel Electrophoresis

SDS Polyacrylamid Gel Electrophoresis

Western Blot

Microbial B₁₂ Plate Assay

Describe the experiments, research and protocols you used in your iGEM project.

What should this page contain?

Protocols
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Revision as of 07:55, 11 October 2016 (view source) To.bi.s (Talk \| contribs) ← Older edit		Revision as of 11:20, 11 October 2016 (view source) To.bi.s (Talk \| contribs) Newer edit →
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	<h3>3. Transformation</h3>		<h3>3. Transformation</h3>

		+	<p>For our transformation experiments, we used electroporation for all our self prepared competent cells. For the heat competent <i>E. coli</i> Top10 cells provided with the pBAD202 expression system, we used the manufacturer's heat shock transformation protocol.</p>

Difference between revisions of "Team:Goettingen/Experiments"

Revision as of 11:20, 11 October 2016

Experiments

Overview of Experiments

1. Production Strains

2. BioBricks

3. Transformation

Protocols

Media

LB Medium

M9 Minimal Medium

Guttman Medium (B₁₂ Assay)

S. blattae Medium Anaerobic

Cultivation and Transformation

Cultivation of Escherichia coli, Shimwellia blattae and Salmonella typhimurium TA 100

Cultivation of Raoultella planticola

Preparation of Electrocompetent Cells

Transformation Electroporation of Electrocompetent Cells

Heat Shock Transformation of Heat Competent Cells

PCR and Cloning

Entry 1

ENtry 2

Entry 3

Entry 4

Analytic Tests

Agarose Gel Electrophoresis

SDS Polyacrylamid Gel Electrophoresis

Western Blot

Microbial B₁₂ Plate Assay

What should this page contain?

Inspiration

Contact

Navigation

Sponsors

Difference between revisions of "Team:Goettingen/Experiments"

Revision as of 11:20, 11 October 2016

Experiments

Overview of Experiments

1. Production Strains

2. BioBricks

3. Transformation

Protocols

Media

LB Medium

M9 Minimal Medium

Guttman Medium (B12 Assay)

S. blattae Medium Anaerobic

Cultivation and Transformation

Cultivation of Escherichia coli, Shimwellia blattae and Salmonella typhimurium TA 100

Cultivation of Raoultella planticola

Preparation of Electrocompetent Cells

Transformation Electroporation of Electrocompetent Cells

Heat Shock Transformation of Heat Competent Cells

PCR and Cloning

Entry 1

ENtry 2

Entry 3

Entry 4

Analytic Tests

Agarose Gel Electrophoresis

SDS Polyacrylamid Gel Electrophoresis

Western Blot

Microbial B12 Plate Assay

What should this page contain?

Inspiration

Contact

Navigation

Sponsors

Guttman Medium (B₁₂ Assay)

Microbial B₁₂ Plate Assay