Team:Uppsala/Experiments
3A Assembly
1. Digestion
A 25 µL master mix was prepared for both of the inserts. The backbone plasmid was already precut, but to cut backbone plasmids EcoRI-HF and PstI would be used (the same volume).
Enzyme Master Mix for the first insert:
| NEB Buffer 2 | 5 µL |
| EcoRI-HF | 0.5 µL |
| SpeI | 0.5 µL |
| Milliq | 19 µL |
4 µL of the master mix was then mixed with 4 µL of the enzyme.
Enzyme Master Mix for the second insert:
| NEB Buffer 2 | 5 µL |
| XBaI | 0.5 µL |
| PstI | 0.5 µL |
| Milliq | 19 µL |
4 µL of the master mix was then mixed with 4 µL of the enzyme.
Both digestion mixtures were digested at 37 °C for 30 minutes. The mixtures were then left at 80 °C for 20 minutes to heat kill the enzymes. These steps were performed in a PCR machine.
2. Ligation
In this step it is important to have equimolar amount of cut plasmid backbone and digest product. In our case the length of everything was very similar, therefore the same volumes could be used, however that can always be done. The total reaction volume is 10 µL
Ligation Mixture:
| Plasmid backbone | 2 µL |
| Enzyme insert cut at E and S | 2 µL |
| Enzyme insert cut at X and P | 2 µL |
| T4 DNA Ligase Buffer | 1 µL |
| T4 DNA Ligase | 0.5 µL |
| MilliQ | 2.5 µL |
The mixture was then ligated at 16 °C for 30 minutes. Another heat kill around at 80 °C for 20 minutes was then performed.
For the transformation step see the Phusion PCR, Gibson Assembly and Transformation protocol.
Phusion PCR, Gibson assembly and Transformation
1. Phusion PCR
Total reaction volume 50 µL
Amount of plasmid per reaction ~30 pg
Phusion PCR reaction mixture:
| Plasmid | …. µL (~30 pg) |
| 5X Phusion HF Buffer | 10 µL |
| dNTP (10 mM) | <1 µL/td> |
| Reverse Primer (10 µM) | 2.5 µL |
| Forward Primer (10 µM) | 2.5 µL |
| Phusion DNA pol | 0.5 µL |
| DMSO | 1.5 µL |
| H2O | up to 50 µL |
| Total volume | 50 µL |
PCR reaction conditions:
| 1) Initial denaturation: | 98 °C for 30 seconds |
| 2) Denaturation in following cycles: | 98 °C for 10 seconds |
| 3) Primer annealing: | Temperature gradient between 58–65 °C for 30 seconds |
| 4) Extension: | 72 °C 1.5 minutes |
| 5) Final DNA synthesis: | 72 °C for 5 minutes |
Steps 2–4: 25 cycles
Lid temperature at 105 °C
2. DpnI Treatment of the PCR Fragment
To the 50 µL PCR reaction mix from step 1 add:
2.4 µL “FastDigest” DpnI
6 µL 10X “FastDigest” Buffer
1) Incubate reaction mixture at 37 °C for 1 hour
2) Purify the DNA in the mixture with “PureLink Quick PCR Purification Kit” (Invitrogen). Milliq was used instead of TE buffer when eluting the DNA from the spin column in the final step of the DNA purification. The optional washing step was skipped
3) Measure the DNA concentration on Nanodrop in units of ng/µL. 2 µL was used for each measurement
4) Store the DNA at -20 °C until further use
3. Gibson Assembly Reaction
1. Mix PCR fragment from the plasmid and the Gibson fragment containing the insert. The total volume is 20 µL:
Mix i) the PCR fragment made from the plasmid and ii) the Gibson fragment containing the gene of interest. Total volume of the Gibson reaction mix is 20 uL :
| Plasmid PCR fragment | .… µL (~0.02 pmol) |
| Gibson fragment | .… µL (~0.06 pmol) |
| Gibson Assembly Master Mix (2x) | 10 µL |
| H2O | up to 20 µL |
| Total volume | 20 µL |
2. Incubate the mixture at 50 °C for 1 hour
4. Transformation
1. Take out competent cells (we used Escherichia Coli TOP10 for cloning and E. coli BL21 (DE3*) for protein expression from Invitrogen) from the -80 °C and thaw them on ice from approximately 15 minutes.
2. Add 2 µL of the Gibson mixture/Ligation mixture to the competent cells or ~7.5 ng of gBlocks.
3. Incubate on ice for 30 minutes.
4. Incubate at 42 °C for 30 seconds (water bath).
5. Incubate on ice for 5 minutes.
6. Add 200 µL of LB to the mixture and incubate it at 37 °C with 200 rpm for 1 hour.
7. Plate the mixtures on chloramphenicol plates (200 µL 1:1 dilution and 200 µL 1:100 diluted with LB). Beads were used to spread the mixture on the plates (10–20 beads).
8. Incubate plates overnight at 37 °C.
Electroporation
1. Grow overnight culture in 5 mL LB media.
2. Wash with 1 mL 10 % glycerol 5 times in cold room.
3. Resuspend in 250 µL 10% glycerol. This is enough for 10 electroporation transformations.
4. Put 1 µL plasmid (~10 ng/µL) in 25 µL cells. Transfer to a cold electroporation cuvette.
5. Electroporate with a 1.8 kV pulse. Immediately add 300 µL SOC media. Incubate in 37°C with 180 rpm shaking for 1h. Plate on appropriate antibiotic agar plate.
Colony PCR and Gel Electrophoresis
1. Colony PCR
Total reaction volume 20 µL.
DreamTaq PCR Reaction Mixture:
| 10X Green DreamTaq Buffer | 2 µL |
| dNTP Mix, 10 mM | 0.4 µL |
| Forward Primer | 0.1–1.0 µM (~0.5 µL) |
| Reverse Primer | 0.1–1.0 µM (~0.5 µL) |
| Template DNA | 10 pg – 1 µg |
| DreamTaq DNA Polymerase | 0.1 µL |
| Milliq | up to 20 µL |
A master mix containing everything put the DNA polymerase was made. Enzyme was then added to each PCR tube.
PCR reaction conditions:
| 1) Initial denaturation: | 95 °C for 3 minutes |
| 2) Denaturation in following cycles : | 95 °C for 30 seconds |
| 3) Primer annealing: | 56 °C for 40 seconds |
| 4) Extension: | 72 °C 1 minute |
| 5) Final DNA synthesis: | 72 °C for 5 minutes |
Steps 2–4: 32 cycles
Lid temperature at 105 °C
2. Gel electrophoresis
Preparation of agarose for gel electrophoresis:
1. Mix 0.5 % TEB with agarose so that the amount of agarose is 0.8 %
2. Microwave the mixture until it starts boiling. DO NOT PUT THE LID ON!
3. Store at 65 °C until further use
Preparation of gels
1. Take 50 mL of agarose per gel into a separate bottle
2. Add 5 µL of SyberSafe per gel into the agarose
3. Pour the agarose into the cassette, put the comb in and let it polymerize for ~30 minutes
4. Put the gel into the electrode chamber and fill it up with 0.5 % TEB buffer
5. Load 12 µL of PCR product into the wells and add 5 µL of 1 kb Generuler
6. Run the gel at 100 V for 30–60 minutes
Expression, IMAC Purification, SDS-PAGE analysis & Activity Measurements
Expression
1. Transform plasmid in BL21 (DE3*) E. coli cells to use for expression.
2. Inoculate three colonies from transformation plate into 50 mL LB with 30 µg/ml chloramphenicol. Make a negative control without any colony and grow overnight.
3.Take 20 mL of culture in 2 L LB with 30 µg/mL chloramphenicol to make expression cultures, divided into two 1 L E-flasks. Grow at 37 °C 110 rpm for about 3 hours until 0.6 < OD600 < 0.8. Take out negative control sample that is not induced. Add 0.25–1 mM IPTG to induce protein expression and grow at 37 °C 110 RPM for about 4–6 hours.
4. Divide culture into 4 tubes and centrifuge culture at 5000 rpm for 30 min. Discard supernatant and resuspend each pellet in about 20 mL LB. Pour over to tw0 50 mL Falcon tubes and centrifuge 5 min at 7000 rpm. Discard the supernatant. Flash freeze pellet in liquid nitrogen and store at -80 °C.
IMAC Purification
Buffer A, 1 L
The buffer was made with MilliQ H2O. NaOH was added until pH 7.4 and sterile filtered on 0.2 µm filter.
| Buffer component | Concentration |
|---|---|
| NaH2PO4/NA2HPO4 | 20 mM |
| NaCL | 500 mM |
| Imidazole | 20 mM |
| beta-ME (added on day of chrom.) | 5 mM |
| Protease inhibitor pill EDTA-free(added on day of chrom.) | 30% of pill |
Buffer B, 0.5 L
The buffer was made with MilliQ H2O. Phosphoric acid was added until pH 7.4 and sterile filtered on 0.2 µm filter.
| Buffer component | Concentration |
|---|---|
| NaH2PO4/NA2HPO4 | 20 mM |
| NaCl | 500 mM |
| Imidazole | 500 mM |
| beta-ME (added on day of chrom.) | 5 mM |
| Protease inhibitor pill (added on day of chrom.) | 30% of pill |
(Day of purification) 1. Thaw the cells (2 pellets) and add 10 mL buffer A to each pellet. Add 20% of EDTA-free protease inhibitor pill to each pellet. Add 500 µL lysozyme (10 mg/ml) and 2 µL DNAse I (238 units/µL). Put on stirring at 4 °C for 40 min.
2. Use a cell disruptor to lyse the cells and pellet for 1 hour at 3800 rfc and run the supernatant through a 0.2 µm vacuum filter.
3. Add the supernatant to Ni2+-agarose column on an ÄKTA protein purification system. 3 mL fractions were collected.
4. Wash the injection tubes by running buffer A for some minutes. Wash column with buffer B and equilibrate with buffer A. Wash with 50–100 mL buffer A until A280 hit baseline. Start eluting gradient to 100 % buffer B in 60 min. Collect 3 mL fractions.
5. SDS-PAGE analysis
a. Run the fractions containing protein on SDS-PAGE to identify fractions holding the desired protein.
b. Load premade gels (4–20 % SDS) with 20 µL sample (10 µL protein sample and 10µL of Laemmli buffer) as well as 6 µL PageRuler-prestained ladder 10-180 kDa (ThermoFisher) and run the gel in running buffer at 200 V for about 30 min.
c. Stain gels in Coomassie blue, destain and document the result.
6. Pool fractions containing purified protein and perform buffer exchange to PBS+500 mM NaCl using Amicon Ultra Centrifugal filters 10 kDa. This should be done before performing activity measurements.
Activity measurements
1. Before activity measurements of the purified enzyme buffer exchange to PBS+500 mM NaCl should be performed using Amicon Ultra Centrifugal filters 10 kDa. 2. The buffers should be freshly prepared. 3. Use plate reader to performed the measurements. 4. The final volume in each well was 75 µL. 65 µL + 10 µL of protein (enzyme). 5. Substrates were dissolved in DMSO stock solution. 6. Negative control (substrate without protein) and positive control (product without protein) were used. 7. Buffer without protein, substrate or product was used as a blank. 8. Absorbance spectra was in the 200–700 nm range and measurements were taken at 1–15 min intervals until activity had been observed.
CsADH2946
Protein concentration was varied in each well, see specifics for the experiment here
| Buffer component | Concentration |
|---|---|
| KCl | 100 mM |
| NAD+ | 0.67 mM |
| 2-Mercaptoethanol | 10 mM |
| Crocetin dialdehyde | 150 µM |
| Crocetin (positive control only) | 150 µM |
| Tris-HCl, pH 8.0 | 100 mM |
λ red recombination
P1 LB medium
Standard LB + 10-25 mM MgCl2, 5 mM CaCl2, 0.1-0.2% glucose
Na-citrate LB medium”
Standard LB + 100 mM Na-Citrate (pH 5.5)
Lysate preparation
1. Grow an overnight culture of the donor strain (with selection if needed).
2. Inoculate 2 x 4 ml P1 LB medium in 50 ml tubes with 50µl culture per tube.
DO NOT ADD ANTIBIOTICS!
Grow shaking with aeration at 37 ˚C for 1-1.3 h. When the cells are in early log phase (slightly turbid, but noticeable growth), add 100 µL of P1 phage lysate to one of the cultures, continue growing at 37 ˚C. Monitor for 1–4 h, and when the culture has lysed, transfer the cells in the other tube to 2 ml tubes, spin down the cells and resuspend them in 200 µl P1 LB. Add the resuspended cells to the lysed tube and continue growing at 37 ˚C. Monitor for 1–4 h until the culture has lysed.
3. Transfer lysate to 2 ml tubes, and add 100 µl of chloroform to the lysate and vortex 10 sec. Centrifuge away the debris (13000 rpm, 2 min) and transfer the supernatant to a fresh tube. Add a few drops of chloroform and store at 4 ˚C.
P1 transduction
1. Grow recipient overnight in P1 LB (1 ml culture per transduction).
2. Add 75 μl donor P1 lysate to 400 μl culture of the recipient strain, mix, and incubate the tubes in a 37 ˚C water bath for 30 min.
6. Spin cells at 6000 rpm for 5 min, remove supernatant carefully and resuspend the pellet in 100 µL Na-citrate LB, vortex well to disperse cells, and plate all of it on an appropriate antibiotic-containing plate.
8. You should get anywhere from ~ 10 to 2000 colonies. These colonies are growing on a plate that is covered with P1 phage, so use a toothpick to touch the top of a few colonies and re-streak on new selective plates for isolated colonies.
9. Test a colony from each re-streak for the presence of the mutant gene you intended to transduce using PCR.
Standard LB + 10-25 mM MgCl2, 5 mM CaCl2, 0.1-0.2% glucose
Na-citrate LB medium”
Standard LB + 100 mM Na-Citrate (pH 5.5)
Lysate preparation
1. Grow an overnight culture of the donor strain (with selection if needed).
2. Inoculate 2 x 4 ml P1 LB medium in 50 ml tubes with 50µl culture per tube.
DO NOT ADD ANTIBIOTICS!
Grow shaking with aeration at 37 ˚C for 1-1.3 h. When the cells are in early log phase (slightly turbid, but noticeable growth), add 100 µL of P1 phage lysate to one of the cultures, continue growing at 37 ˚C. Monitor for 1–4 h, and when the culture has lysed, transfer the cells in the other tube to 2 ml tubes, spin down the cells and resuspend them in 200 µl P1 LB. Add the resuspended cells to the lysed tube and continue growing at 37 ˚C. Monitor for 1–4 h until the culture has lysed.
3. Transfer lysate to 2 ml tubes, and add 100 µl of chloroform to the lysate and vortex 10 sec. Centrifuge away the debris (13000 rpm, 2 min) and transfer the supernatant to a fresh tube. Add a few drops of chloroform and store at 4 ˚C.
P1 transduction
1. Grow recipient overnight in P1 LB (1 ml culture per transduction).
2. Add 75 μl donor P1 lysate to 400 μl culture of the recipient strain, mix, and incubate the tubes in a 37 ˚C water bath for 30 min.
6. Spin cells at 6000 rpm for 5 min, remove supernatant carefully and resuspend the pellet in 100 µL Na-citrate LB, vortex well to disperse cells, and plate all of it on an appropriate antibiotic-containing plate.
8. You should get anywhere from ~ 10 to 2000 colonies. These colonies are growing on a plate that is covered with P1 phage, so use a toothpick to touch the top of a few colonies and re-streak on new selective plates for isolated colonies.
9. Test a colony from each re-streak for the presence of the mutant gene you intended to transduce using PCR.
P1 transduction
E. coli:
DA24100 Escherichia coli K12 MG1655 /pSIM5-Tet Lambda Red system TetR This strain carries the pSIM5-tet plasmid, see above. Grow at 30°C, 12.5 μg/ml tetracycline.
1. Grow strain O/N at 30°C in Salt Free LB (with appropriate antibiotic).
2. Dilute 1:100 in 30°C pre-warmed Salt Free LB (with appropriate antibiotic for pSIM5/pSIM5-Tet/pSIM6/pSIM-ctx) in a 500ml E-flask. 50 ml is enough for 4-8 electroporations.
3. Grow at 30°C shaking until OD600 ≈ 0.3-0.4 (1.5~2 hrs).
4. To induce red expression, move the flask to a 42°C shaking waterbath, incubate with slow shaking (110 rpm) for 15 minutes.
5. Cool cells on ice for at least 10 minutes. Swirl carefully to cool cells.
6. Transfer cells to cold 50 ml tubes and spin down the cells (4500 rpm, 8 min., 4°C).
7. Remove as much as possible of the supernatant and resuspend the cells in 100 µl ice cold sterile water using a 10 µl loop. Add 12 ml ice cold water and pipette up and down a few times (using the pipette-boy). Do not vortex! Pellet the cells at 4500 rpm, 6-10 min, 4°C and repeat the wash one more time.
8. Resuspend the cells in ice-cold 10% glycerol (as small volume as you need, ~300 µl).
9. Mix 40 μl cells and DNA in an electroporation cuvette on ice (2-8 µl de-salted PCR product or up to half of what you get from a normal 20 μl PCR reaction, 100-500 ng.
10. Electroporate (2.5 kV 200 Ω, 25 µF). Immediately resuspend the cells in 1 ml 42°C SOC or SOB and transfer to 10 ml plastic tubes.
11. Optional: Incubate the cells in a 42°C water bath for 10 minutes.
12. Let the cells recover for at least 3 hrs or overnight in a 30°C shaking incubator.
13. Plate on appropriate selective medium, incubate the plates at 30°C O/N.
14. Restreak colonies on selective medium at 42°C. Test for loss of the plasmid. (Consider freezing a clone that still contains the pSIM-plasmid.)
Extracting zeaxanthin from E. coli strain and TLC
1. Pick a colony and grow in LB overnight in 30°C in shaking incubator.
2. For an analytical extraction, spin down 10 mL cells and lyse with 1 mL acetic acid.
3. Pour 1 mL toluen and 1 mL H2O into the tube to create a two-phase separation. Spin down. Take the upper organic phase into a new tube and evaporate under vacuum until a yellow pigment powder is visible. Resolve in 10 uL toluen and measure the absorbance spectra on nanodrop.
4. Run thin layer chromatography (TLC) on a silica coated aluminum plate with 90% chloroform and 10% ethanol as mobile phase.
E. coli:
DA24100 Escherichia coli K12 MG1655 /pSIM5-Tet Lambda Red system TetR This strain carries the pSIM5-tet plasmid, see above. Grow at 30°C, 12.5 μg/ml tetracycline.
1. Grow strain O/N at 30°C in Salt Free LB (with appropriate antibiotic).
2. Dilute 1:100 in 30°C pre-warmed Salt Free LB (with appropriate antibiotic for pSIM5/pSIM5-Tet/pSIM6/pSIM-ctx) in a 500ml E-flask. 50 ml is enough for 4-8 electroporations.
3. Grow at 30°C shaking until OD600 ≈ 0.3-0.4 (1.5~2 hrs).
4. To induce red expression, move the flask to a 42°C shaking waterbath, incubate with slow shaking (110 rpm) for 15 minutes.
5. Cool cells on ice for at least 10 minutes. Swirl carefully to cool cells.
6. Transfer cells to cold 50 ml tubes and spin down the cells (4500 rpm, 8 min., 4°C).
7. Remove as much as possible of the supernatant and resuspend the cells in 100 µl ice cold sterile water using a 10 µl loop. Add 12 ml ice cold water and pipette up and down a few times (using the pipette-boy). Do not vortex! Pellet the cells at 4500 rpm, 6-10 min, 4°C and repeat the wash one more time.
8. Resuspend the cells in ice-cold 10% glycerol (as small volume as you need, ~300 µl).
9. Mix 40 μl cells and DNA in an electroporation cuvette on ice (2-8 µl de-salted PCR product or up to half of what you get from a normal 20 μl PCR reaction, 100-500 ng.
10. Electroporate (2.5 kV 200 Ω, 25 µF). Immediately resuspend the cells in 1 ml 42°C SOC or SOB and transfer to 10 ml plastic tubes.
11. Optional: Incubate the cells in a 42°C water bath for 10 minutes.
12. Let the cells recover for at least 3 hrs or overnight in a 30°C shaking incubator.
13. Plate on appropriate selective medium, incubate the plates at 30°C O/N.
14. Restreak colonies on selective medium at 42°C. Test for loss of the plasmid. (Consider freezing a clone that still contains the pSIM-plasmid.)
Extracting zeaxanthin from E. coli strain and TLC
1. Pick a colony and grow in LB overnight in 30°C in shaking incubator.
2. For an analytical extraction, spin down 10 mL cells and lyse with 1 mL acetic acid.
3. Pour 1 mL toluen and 1 mL H2O into the tube to create a two-phase separation. Spin down. Take the upper organic phase into a new tube and evaporate under vacuum until a yellow pigment powder is visible. Resolve in 10 uL toluen and measure the absorbance spectra on nanodrop.
4. Run thin layer chromatography (TLC) on a silica coated aluminum plate with 90% chloroform and 10% ethanol as mobile phase.
