Protocols

Here you can find all the protocols followed during the development of each of our subprojects.

• Correct folding study of target protein
• Structure analysis of our targets and their interactions
• Investigation of P3 threshold for E. coli resistance
• Conversion of BACTH into an iGEM standard and analysis of function
• Set up of flow system

Correct folding study of target protein

Transformation of E. coli

Before starting

1. Chill ddH₂O on ice for at least 2 hours.
2. Chill 1 mm gap electroporation cuvettes.
3. Cool down a cooling centrifuge to 2 °C.

Set up culture

1. Set up 2 lid-punctured reaction tubes containing 1.4 mL of fresh LB medium supplemented with chloramphenicol (10 µg/mL).
2. Inoculate each with 40 µL of the overnight culture.
3. Incubate the tubes in the thermoshaker at 37 °C at 950 rpm for 1.5 hours.

Making cells electrocompetent

1. The day before start a 1 mL LB culture with the respective antibiotic when necessary and incubate at 30 °C (e.g. with pSC101BADαβγA) or 37 °C with 950 rpm overnight.
2. Place a bottle of sterile water at least for 2 hours on ice.
3. Start a 1.4 mL LB culture with the respective antibiotic, when necessary with 30 µL of the overnight culture and incubate at 30 °C (e.g. with pSC101BADαβγA) or 37 °C with 950 for 2 hours.
4. Put the cells on ice and keep them on ice whenever possible.
5. Spin the cells down at 8,000 rpm for 30 seconds in a cooling centrifuge at 2 °C.
6. Discard the supernatant.
7. Resuspend the pellet in 1 mL of ice cold water with a pipette.
8. Spin the cells down at 8,400 rpm for 30 seconds at 2 °C.
9. Discard the supernatant.
10. Resuspend the pellet in 1 mL of ice cold water with a pipette.
11. Spin the cells down at 8,800 rpm for 30 seconds at 2 °C.
12. Discard as much supernatant as possible.
13. The remaining solution should be about 30 µL, resuspend the cells in it.
14. Use the cells immediately.

Electroporation

1. Add 2 µL of plasmid to the resuspended cells in one reaction tube, and pipette the mixture into the chilled electroporation cuvette, the remaining cells are the back-up.
2. Electroporate at 1,350 V, 10 µF, 600 ohms.
3. Add 1 mL LB medium without antibiotics to the cuvette. Mix the cells carefully by pipetting up and down and pipette back into the reaction tube. Incubate the cultures at 30 °C with shaking for 60 minutes.

Plating

1. Plate 100 µL of the cells with a loop on LB agar plates (with necessary antibiotics if needed) and incubate them overnight.

Restriction digestion

Master mix for digestion of the plasmid DNA:

• (n+1) x 10 µL of 10x CutSmart buffer.
• (n+1) x enzyme depending on the activity, [in our case NheI-HF: 1µL and NotI-HF: 1µL].
• (n+1) x 4.8 µL ddH₂O.
• (n+1) x 6.0 µL.

Add 4 µL of plasmid to each reaction tube. Mix together and incubate for 1 to 2 hours at 37 °C.

Ligation

Mix the following components and incubate overnight at 16 °C:

• 10 µL vector (pET28a).
• 1 µL insert (HER).
• 2 µL T4 ligase buffer.
• 1 µL T4 DNA ligase.
• 6 µL ddH₂O.

Add 4 µL of plasmid to each reaction tube. Mix together and incubate for 1 to 2 hours at 37 °C.

Protein extraction and purification

Mix the following components and incubate overnight at 16 °C.

1. Set up 10 mL culture per 1 L of main culture (in this case LB with antibiotics- Kanamycin(Kan)).
2. Transfer the 10 mL culture to 1 L LB with antibiotics (Kan) [Day 1].
3. Grow the culture to an optical density of 0.6-0.8 at λ=600 at 37 °C (~3 hours).
4. Remove 1 mL of culture for analysis by SDS-PAGE (pre-induced sample) and another 1 mL for preparation of a glycerol stock (add 500 µL of 50 % glycerol to 500 µL of culture).
5. Induce expression of the remaining culture by adding 500 µL of 1 M IPTG for 1 L of culture. Incubate overnight at 30 °C [Day 2].
6. Transfer 300 µL of the induced culture to a microcentrifuge tube and collect the cells by centrifugation at 10 kg for 1 minutes. Discard the supernatant and resuspend the pellet in Lamly with ME for SDS-PAGE (induced sample).
7. Centrifuge the remaining culture in 1 L tubes at 3000 g for 20 minutes at 4 °C.
8. Wash the pellet with PBS and pool everything together. Vortex it, centrifuge it shortly and remove PBS.
9. Resuspend the pellet in 50 mL lysis buffer with inhibitors.
10. Lyse the cells by EMULSIFLEX homogenisator (it is similar to a French press, breaks cells by mechanical force).
11. Centrifuge the suspension at 45,000 rpm, 30 minutes at 4 °C.
12. Take the supernatant for IMAC affinity chromatography (remove 200 µL of supernatant for analysis by SDS-PAGE and also 100 µL of pellet for SDS-PAGE).
13. IMAC by His-tag:
1. Equilibrate column with buffer.
2. Load lysate on the column (1h). Take 100 µL for analysis by SDS-PAGE.
3. Wash column.
4. Elute with elution buffer (30 minutes).
5. Take the correct elutes and discard the rest.
6. If elutes are of the same peak, pool them together and take 50 µL for analysis by SDS-PAGE.
14. This elute was further subjected to size exclusion chromatography and the same procedure as above was repeated.

Staining protocol for Blue Native PAGE

1. Take out the gel and place it in ~100 mL fixing solution and cook it in a microwave for 45 seconds at high (950 to 1,100 watts).
2. Shake the gel on an orbital shaker for 15 minutes. Pour off the fixing solution and pour another 100 mL of fixing solution.
3. Redo the first two steps again except for shaking which is for 20 minutes this time.
4. Take out the fixing solution and put in the staining solution (~100 mL) and cook for 45 seconds.
5. Shake it on a shaker for 30 minutes.
6. Pour off the solution and add ~100 mL destaining solution to it and cook for 2 minutes.
7. Shake it on a shaker for 30 minutes. Some bands might already be visible.
8. Wash with ddH₂O three times. Pour in ddH₂O and shake it on a shaker overnight after which imaging can be done.

Circular dichroism

• CD experiment was done using Chirascan CD spectrometer.
• The protocol followed is described in the following paper by Greenfield (2006) Using circular dichroism spectra to estimate protein secondary structure. More information was obtained Protein Structure and Conformational Studies.

Buffers used

• Lysis buffer (0.5 L): 50 mM Tris-HCl at pH 8, 130 mM KCl, 10mM KCl, 10 mM imidazole, 5 mM MgCl₂, 1 mM PMSF, comprete protease inhibitor cocktail (Roche), 20 mM MgSO₄.
• Equilibrium buffer for His tag chromatography (0.5 L): 50 mM Tris-HCl at pH 8, 130 mM KCl, 10 mM imidazole, 5 mM MgCl₂.
• Elution buffer for His tag chromatography (0.1 L): 50 mM Tris-HCl at pH 8, 130 mM KCl, 10 mM imidazole, 5 mM MgCl₂.
• Equilibrium buffer for size exclusion chromatography (0.5 L): 50 mM Tris-HCl at pH 8, 130 mM KCl, 5 mM MgCl₂.
• Exchange buffer for dyalisis (5 L): 20 mM sodium dihydrogen phosphate, 50 mM sodium tetraborate, 20 mM sodium fluoride.

Structure analysis of our targets and their interactions

For the protocols followed in this suproject, please refer to the Methods.

Investigation of P3 threshold for E. coli resistance

Transformation of E. coli

Before starting

1. Chill ddH₂O on ice for at least 2 hours.
2. Chill 1 mm gap electroporation cuvettes.
3. Cool down a cooling centrifuge to 2 °C.

Set up culture

1. Set up 2 lid-punctured reaction tubes containing 1.4 mL of fresh LB medium supplemented with chloramphenicol (10 µg/mL).
2. Inoculate each with 40 µL of the overnight culture.
3. Incubate the tubes in the thermoshaker at 37 °C at 950 rpm for 1.5 hours.

Making cells electrocompetent

1. The day before start a 1 mL LB culture with the respective antibiotic when necessary and incubate at 30 °C (e.g. with pSC101BADαβγA) or 37 °C with 950 rpm overnight.
2. Place a bottle of sterile water at least for 2 hours on ice.
3. Start a 1.4 mL LB culture with the respective antibiotic, when necessary with 30 µL of the overnight culture and incubate at 30 °C (e.g. with pSC101BADαβγA) or 37 °C with 950 for 2 hours.
4. Put the cells on ice and keep them on ice whenever possible.
5. Spin the cells down at 8,000 rpm for 30 seconds in a cooling centrifuge at 2 °C.
6. Discard the supernatant.
7. Resuspend the pellet in 1 mL of ice cold water with a pipette.
8. Spin the cells down at 8,400 rpm for 30 seconds at 2 °C.
9. Discard the supernatant.
10. Resuspend the pellet in 1 mL of ice cold water with a pipette.
11. Spin the cells down at 8,800 rpm for 30 seconds at 2 °C.
12. Discard as much supernatant as possible.
13. The remaining solution should be about 30 µL, resuspend the cells in it.
14. Use the cells immediately.

Electroporation

1. Add 2 µL of plasmid to the resuspended cells in one reaction tube, and pipette the mixture into the chilled electroporation cuvette, the remaining cells are the back-up.
2. Electroporate at 1,350 V, 10 µF, 600 ohms.
3. Add 1 mL LB medium without antibiotics to the cuvette. Mix the cells carefully by pipetting up and down and pipette back into the reaction tube. Incubate the cultures at 30 °C with shaking for 60 minutes.

Plating

1. Plate 100 µL of the cells with a loop on LB agar plates (with necessary antibiotics if needed) and incubate them overnight.

Restriction digestion

Master mix for digestion of the plasmid DNA:

• (n+1) x 10 µL of 10x CutSmart buffer.
• (n+1) x enzyme depending on the activity, [in our case NheI-HF: 1µL and NotI-HF: 1µL].
• (n+1) x 4.8 µL ddH₂O.
• (n+1) x 6.0 µL.

Add 4 µL of plasmid to each reaction tube. Mix together and incubate for 1 to 2 hours at 37 °C.

Ligation

Mix the following components and incubate overnight at 16 °C:

• 10 µL vector (pET28a).
• 1 µL insert (HER).
• 2 µL T4 ligase buffer.
• 1 µL T4 DNA ligase.
• 6 µL ddH₂O.

Add 4 µL of plasmid to each reaction tube. Mix together and incubate for 1 to 2 hours at 37 °C.

Top agar for blue-white screening

• F+ strains were grown overnight.
• 3 mL of top agar (0.7 %) was prepared with IPTG (0.1 mM final) and X-Gal (40 µg/mL final).
• Top agar was hold warm at 42 °C.
• 200 µL of overnight culture was added.
• Phages were added.
• LB plate was overlayed with the prepared top agar.

Measurement of the expression of P3 by E. coli co-expressed with CFP

• Two cell cultures were cultivated at 37 ° C and at 200 rpm in a shaking incubator for 24 hours.
• The optical density (OD) was measured until it had a value of 1.4 for both cultures.
• To induce the CFP expression IPTG was added to one of the cultures to a final concentration of 2 mM.
• After four hours of induction time samples were taken from the cultures and analyzed in a fluorescence spectrometer
• The CFP fluorescence signal of the induced culture should be compared with the signal of the IPTG free culture. CFP can be excited at 439 nm and emits fluorescent light with a wavelength of 476 nm (BBa_E0020).
• For the CFP detection both culture samples were set to an OD₆₀₀ of 1 and measured in the fluorescence spectrometer with the excitation varying from 350 to 450 nm (in steps of 5 nm) and the emission varying from 400 to 500 nm (in steps of 5 nm) leading to a 3D scan of the E. coli fluorescence.

Analysis of the plasmid stability

• Take samples every 3 hours together with the ones for the phage infection.
• Dilute the samples to a concentration of 10^-5 and first plated on a LB plate without chloramphenicol and grown overnight at 37 °C.
• Count the colonies on the next they and transfer them with a stamp to a LB-plate containing chloramphenicol and grown overnight at 37 °C.
• Count the colonies aagain on the next day. Due to the fact that only the colonies containing the plasmid with the antibiotic resistance are able to grow, a ratio of stable cells can be calculated.

Analysis of the phage infection

• Use the 10^-5 dilution from the plasmid stability to analyze if the phages are washed out during the continuous cultivation and infection in the lagoon.
• Plate 100 μL of the sample on a LB-plate containing chloramphenicol and X-Gal.
• The infected colonies will turn blue because the phages carry a gen for the galactosidase leading to a blue color production..

Conversion of BACTH into an iGEM standard and analysis of function

Set up of flow system

Continuous stirred-tank cultivation

• Fill the bioreactor (1 L Applikon) with 300 mL minimal medium with an OD₆₀₀ of 0.04.
• Inoculate the bioreactor with the medium and stir at 350 rpm and aerate with 5 NL h^-1 (synthetic air).
• Grow the culture until it reaches an OD₆₀₀ of 0.2.
• Start the continuous cultivation by activating the feed pump to supply the reactor with fresh medium (62.5 mL h^-1).
• The culture is continuously removed from the bioreactor and pumped to the lagoon.
• The medium in the bioreactor is set to 250 mL by adjusting the height of the extraction port. With the same principle the volume in the lagoon was set to 63 mL.
• The culture in the bioreactor has to pass 2-3 volume changes before it reaches the steady state. In the next step the phages are added to the lagoon.
• To ensure that the phages have enough time to regenerate and reproduce, they have 1 hour (1 volume change in the lagoon) without any influence from the inducer.
• Isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to the feed entering the lagoon (IPTG feed 6 mL h^-1; final IPTG concentration: 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM).
• Increase the IPTG concentration every 3 hours to ensure that the E. coli have at least 1 volume change with the desired IPTG concentration.

Biobrick assembly

1. Restriction digestion of plasmids parts (P3, HER2, T25, T18, ZHer2, LZT18, LZT25) from IDT (Parts) using EcoRI and PstI and pSB1C3 iGEM biobrick plasmid backbone.
2. Running gel electrophoresis of digested parts (P3, HER2, T25, T18) on 0.7 % gel and ZHER2, LZT18 on 2 % agarose gel.
3. Gel purification of parts.
4. Ligation of parts into digested pSB1C3 backbone.
5. Transforming E. coli GB05 with ligated plasmids.
6. Checking for transformation efficiency and subculturing of clones.
7. Plasmid preparation from transformed clones.
8. Sequencing of plasmids.

Standard protocols

1. HiYield Presto Mini Plasmid Kit.
2. HiYield Gel/PCR DNA Extraction Kit.
3. How to make cells competent for electroporation.
4. Electroporation.
5. Preparation of 0.7% and 2% agarose gel.
6. DNA precipitation.
7. Antibiotics.