Team:UESTC-China/protocal

Team:UESTC-China/Protocol - 2017.igem.org

Bacteria

1. Competent E.coli Cells Preparation

  1. Inoculate E.coli strains on culture plate without antibiotics and streak, place upside down and culture at constant temperature of 37°C overnight.
  2. Select a well-grew colony using a sterile toothpick and inoculate it into 20ml LB medium with no antibiotics, culture at 37°C overnight while shaking (180rmp).
  3. Pipet 1ml bacterium solution into 100ml LB liquid medium without antibiotics, incubate at 37°C for 1 hour and 40 minutes while shaking, at 180rmp, making OD600 is 0.4—0.6.
  4. Transfer the culture solution into a 50ml BD tube, incubate on ice for 10 minutes.
  5. Precool the centrifuge in advance and centrifugation at 4°C, 4000rmp for 10 minutes.
  6. Discard the supernatant, add 16ml pre-cooled 0.1M CaCl2 into a centrifuge tube, pellet cells and re-suspend, then incubate on ice for 10 minutes, and centrifugation at 4°C, 4000rmp for 10 minutes.
  7. Discard the supernatant, add 1.5ml pre-cooled 0.1M CaCl2 with 15% glycerol, re-suspend and incubate on ice for 10 minutes.
  8. Add aforesaid suspensions into pre-cooled 1.5ml centrifuge tubes with 100ul per tube, freeze at -80°C.

2. Overnight Cultures

  1. Add 6mL autoclaved LB in a 10ml EP bottle.
  2. Pipet 6μL of 1000X antibiotic into the LB.
  3. Select a single colony using a sterile toothpick or pipette tip.
  4. Place toothpick or pipette tip in the culture tube and stir.
  5. Remove toothpick, or leave in the bottle.
  6. Place culture tube in incubator at 37°C overnight shaking vigorously (220 RPM).

3. Plates Screening

  1. Pour 20mL autoclaved LB with appropriate antibiotic into each plate.
  2. Take the appropriate amount of bacterium suspension on the plate and spread it homogeneous.
  3. Seal the plate with parafilm, place upside down and culture at constant temperature of 37°C for 12 – 14 hours.

4. Bacteria Preservation

  1. Mix 600ul with 600ul 40% glycerol
  2. Store at -20℃

Molecular Biotechnology

We used different molecular biological methods in our project. All used methods are listed below.

1. PCR

In our project, in order to be able to finally amplify the target fragments. We try the following PCR systems and the procedure settings

KOD amplification System

Component 50ul Reaction
10× KOD buffer 5μl
2mM dNTPs 5μl
25mM MgSO4 3μl
10μM forward primer 1μl
10μM Reverse primer 1μl
Temp 1μl(1ng/μl)
KOD-Plus-Neo 1μl
ddH2O 33μl

KOD PCR Program

Segment Cycles Temperature Time
Initial denaturation 1 94°C 3min
Denaturation 37 94°C 30s
Renaturation 37 56°C 30s
Prolongation 37 68°C 30s/kb
Terminal prolongation 1 68°C 3min
heat preservation 1 12°C 10min

Fusion PCR System

Component 50L Reaction
10× KOD buffer 5μl
2mM dNTPs 5μl
25mM MgSO4 3μl
Forward primer 1μl
Reverse primer 1μl
fragment 1 the moles rate of fragment1 to fragment2 is 1:1
fragment 2 the moles rate of fragment1 to fragment2 is 1:1
KOD enzyme 1μl
ddH2O add to 50μl

Fusion PCR Program

Segment Cycles Temperature Time
Initial denaturation 1 94°C 3min
Denaturation 37 94°C 30s
Renaturation 37 56°C 30s
Prolongation 37 68°C 30s/kb
Terminal prolongation 1 68°C 3min
heat preservation 1 12°C 10min

Colony PCR MasterMix

Component 25ul Reaction
10× Taq buffer 2.5μl
10mM dNTPs 0.5μl
10uM Primer 1 0.5μl
10uM Primer 2 0.5μl
Temp 5μl
Taq enzyme 0.4μl
ddH2O 15.6μl

Colony PCR Program

Segment Cycles Temperature Time
Initial denaturation 1 95°C 5min
Denaturation 37 95°C 30s
Renaturation 37 56°C 30s
Prolongation 37 72°C 40s/kb
Terminal prolongation 1 72°C 5min
heat preservation 1 12°C 10min

2. Agarose Gel

Agarose Gel Preparation

  1. Weight 0.3g agarose, mix with 30ml 1x TAE in a conical flask (0.5g, 50ml for long dies).
  2. Heat the flask in the microwave oven until the solution becomes transparent, cool down to 40°C.
  3. Add 1.5μl EB into solution and mix evenly by shaking slightly (2μl EB for 50ml TAE)
  4. Pour the solution into a die and wait for the gel solidifying.

Agarose Gel Electrophoresis

  1. Add 5μl (10μl) loading buffer into a 25μl (50μl) PCR product.
  2. Apply onto agarose gel together with makers.
  3. Run at 130V for 25 minutes for a full gel.

Gel Extraction of DNA (AXYGEN Gel Extraction Kit)

  1. Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove residual buffer. Transfer the gel slice to a piece or plastic wrap or a weighing boat. Mince the gel into small pieces and weigh. 100 mg of gel is equivalent to a 100 μl volume. Transfer the gel slice into a 1.5 ml microfuge tube.
  2. Add a 3x sample volume of Buffer DE-A.
  3. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75°C until the gel is completely dissolved. Intermittent vortexing to accelerate gel solubilization.
  4. Add 0.5x Buffer DE-A volume of Buffer DE-B, mix. If the DNA fragment is less than 400 bp, supplement further with a 1x sample volume of isopropanol.
  5. Place a Miniprep column into a 2 ml microfuge tube. Transfer the solubilized agarosefrom Step 4 into the column. Centrifuge at 12,000xg for 1 minute.
  6. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 500 μl of Buffer W1. Centrifuge at 12,000xg for 30 seconds.
  7. Discard the filtrate from the 2 ml microfuge tube. Return the Miniprep column to the 2 ml microfuge tube and add 700 μl of Buffer W2. Centrifuge at 12,000xg for 30 seconds.
  8. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Add a second 700 μl aliquot of Buffer W2 and centrifuge at 12,000xg for 1 minute.
  9. Discard the filtrate from the 2 ml microfuge tube. Place the Miniprep column back into the 2 ml microfuge tube. Centrifuge at 12,000xg for 1 minute.
  10. Transfer the Miniprep column into a clean 1.5 ml microfuge tube (provided). To elute the DNA, add 20 μl of deionized water（65℃） to the center of the membrane. Let it stand for 1 minute at room temperature. Centrifuge at 12,000xg for 1 minute.

3. Ligation

T4 ligation System

Component 20ul Reaction
10× T4 Ligation buffer 2μl
T4 Ligation enzyme 1μl
Fragment of digestion the moles rate of insert DNA to vector DNA is 5:1 to 10:1
Vector of digestion the moles rate of insert DNA to vector DNA is 5:1 to 10:1
ddH2O add to 20μl

Incubate at 22℃ for 2 hour

Gibson Assembly

Components 20ul Reaction
20ul Reaction 15μl
Insert DNA the moles rate of insert DNA to vector DNA is 5:1 to 10:1
Vector the moles rate of insert DNA to vector DNA is 5:1 to 10:1
ddH2O add to 20μl

Incubate at 50℃ for 1 hour

Golden Gate System

Components 20ul Reaction
10× T4 Ligation buffer 2μl
BsaⅠ enzyme the moles rate of insert DNA to vector DNA is 5:1 to 10:1
T4 Ligation enzyme the moles rate of insert DNA to vector DNA is 5:1 to 10:1
Every parts add more than 100ng
ddH2O add to 20μl

Colony PCR Program

Segment Cycles Temperature Time
BsaⅠ digestion 10 37°C 5min
T4 ligation 10 16°C 10min
Terminal prolongation 1 37°C 10min
Enzyme inactivation 1 85°C 10min
Heat preservation 1 12°C 10min

4. Transformation

Escherichia coli Transformation

  1. Pipet each ligation product into a 1.5 mL tube with 100uL competent E.coli Dh5α/MC1061
  2. Put on ice for 30 minutes
  3. Incubate at 42°C for 70 seconds
  4. Put it back on ice for 3 minutes
  5. Add 500L LB media
  6. Incubate for 1 hour at 37°C and 180 rpm
  7. Centrifuge tubes for 1 minutes at 8000 rpm
  8. Pipet 500μl supernatants out , pipet up and down the left liquid to suspend the cells
  9. Spread plate with glass bead
  10. Culture in an incubator overnight (18 hours) at 37°C

Agrobacterium Transformation

  1. Have 0.5~1 ng plasmid DNA into 100 µl competent cells, on ice for 30 min
  2. Frozen in liquid nitrogen for 5 min, grow at 37 ℃ for 5 min, on ice for 2 min
  3. Pour in 1000µl LB, 200 rpm, grow at 28℃ for 2~3 h
  4. 6000rpm，2min. Suspend collected bacteria with 100µl LB and evenly coat that at a LB medium. (50 mg/L Rif, and 50 mg/L Kan included)
  5. Grow upside down at 28°C, for 48h
  6. Pick positive clones and grow them in LB medium with antibiotic at 28°C for 48h
  7. Inject to LB medium in flasks by the day of transformation, in the rate of 1:50. Grow to OD600 = 0.5. Ready to infect tobacco leaf discs

5. Plasmids extraction

  1. Collect 5 ml of overnight LB culture. Centrifuge at 12,000×g for 1 minute to pellet the bacteria.
  2. Resuspend the bacterial pellet in 250 μl of Buffer S1 by vortexing.
  3. Add 250 μl of Buffer S2, and mix by gently inverting the tube for 4-6×.
  4. Add 350 μl of Buffer S3, and mix by gently inverting 6-8×. Centrifuge at 12,000×g for 10 minutes to clarify the lysate.
  5. Place a Miniprep column into an uncapped 2 ml Microfuge tube. Transfer the clarified supernatant from Step 4 into the Miniprep column. Transfer the Miniprep column and 2 ml Microfuge tube to microcentrifuge and spin at 12,000×g for 1 minute.
  6. Pipette 500 μl of Buffer W1 into each Miniprep column. Centrifuge at 12,000×g for 1 minute.
  7. Pipette 700 μl of Buffer W2 into each Miniprep column. Centrifuge at 12,000×g for 1 minute.
  8. Discard the filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Add 700 μl of Buffer W2 to the Miniprep column again and centrifuge at 12,000×g for 1 minute.
  9. Discard filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Centrifuge at 12,000×g for 1 minute.
  10. Transfer the Miniprep column into a clean 1.5 ml Microfuge tube. Add 50μl deionized water（65℃）to the center of the membrane. Let it stand for 1 min at room temperature. Centrifuge at 12,000×g for 1 minute.

Plants

Agrobacterium-mediated Tobacco Transformation

  1. Leaf sections ( 1.0 to 1.5 cm² ) aseptically excised fr old tobacco plants were immersed for 10 min in a suspension of A. tumefaciens (piGEM001、002、003、004、005) in MSB-L medium.
  2. Infected leaf sections were blotted dry on a sterile filter paper and transferred onto the basal MSB-Co medium(basal MSB agar with 0.1mg/L a-naphthalene acetic acid (NAA) and 2.0 mg/L 6-benzylaminopurine (6-BA) )for a 2-day co-cultivation at 25℃ in dark.
  3. The sections were then placed on the Agrobacterium-eliminating MSB-S medium (basal MSB agar with 0.1 mg/L NAA, 2.0mg/L 6-BA, 500 mg/L Cef, and 100 mg/L Kan) for 11 days to initiate callus and plantlets formation and get rid of A. tumefaciens at 25℃ 16hr/8hr.
  4. The selection of transformed plantlets was performed by their subculturing in two steps on the same medium.
  5. After the selection, the plantlets were placed on the Agrobacterium-eliminating MSB-R medium (basal MSB agar with 0.1 mg/L NAA, 500 mg/L Cef, and 50 mg/L Kan) to take root and grow up.

Positive Detection of Transgenic Plants

1. Plant Genomic DNA Extraction

  1. Prepare a set of 1.5ml microfuge tubes. Add two or three steel balls in each tubes. CTAB buffer should be incubated at 65℃ ahead of time.
  2. Cut the plant tissue and put it into tubes. Place all tubes in liquid nitrogen and smash it thoroughly.
  3. Add 600μl CTAB into each tube.
  4. Incubate the CTAB/plant extract mixture for about 30 min at 65℃ in a recirculating water bath.
  5. After incubation, add 600μl of trichloromethane into each tube. Mix the solution by inversion. After mixing, spin the mixture at 9000 g for 10 min to spin down cell debris. Transfer the supernatant to clean microfuge tubes.
  6. To each tube add 600μl of isopropyl alcohol. Refrigerate the mixture at -20℃ for 1h.
  7. After refrigerating, centrifuge all tubes at 12000 g for 10 min and pour out the supernatant.
  8. To each tube add 600μl of 75% ethyl alcohol. Spin the mixture at 12000 g for 10 min to precipitate the DNA. Then pour out the supernatant. Generally the DNA can be seen to precipitate out of solution.
  9. Repeat the step 8. Air the liquid in tubes and add 100μl ddH2O to each tube.
  10. Take 1μg genomic DNA extracted to do PCR amplification directly.

2. RT-PCR

Reverse transcription

  1. Prepare the reverse transcription reaction mix by adding the following components in a RNase-Free 1.5 ml microcentrifuge tube on ice.
    • 1μg total RNA
    • 2 μl Random
    • 2 μl Super pure dNTPs (2.5 mM each)
    • Add RNase-Free ddH2O to make total of 14.5 μl
  2. Heat at 70°C for 5 min and then cool on ice immediately for 2 min. Briefly centrifuge and add the following components.
    • 4 μl 5× First-Strand Buffer (including DTT)
    • 0.5 μl RNasin
  3. Add 1 μl (200 U) TIANScript M-MLV, mix gently by pipetting. Incubate the microcentrifuge tube at 25°C for 10 min.
  4. Incubate at 42°C for 50 min.
  5. Heat the sample at 95°C for 5 min to inactivate the TIANScript M-MLV, and then place on ice for downstream experiments or store at -20°C immediately.
  6. Dilute the reaction to 50 μl with RNase-Free ddH2O. Take 3μl for PCR amplification directly.

Amplification System

Components 20ul Reaction
10× Taq buffer 2.5μl
2.5mM DNTPs 2μl
Primer1 0.5μl
Primer2 0.5μl
cDNA (synthesis reaction mixture) 3μl
Taq DNA Polymerase (2.5 U/µl) 0.4μl
ddH2O 16.1μl

PCR Program

Segment Cycles Temperature Time
Initial denaturation 1 95°C 5 min
Denaturation 38 95°C 30 s
Renaturation 38 56°C 30 s
Prolongation 38 72°C 40 s/kb
Terminal prolongation 1 72°C 5 min
Heat preservation 1 12°C 10 min

Note: If the temp is positive control, the volume is 1μl and the cycle of PCR is only 20.

Agarose Gel

The details of Agarose Gel setting are in NO.2 Agarose Gel, section Molecular Biotechnology.

Enzyme Assay

1. Plant Genomic DNA Extraction

Reaction proceeded in 200 mM Tris-SO4 buffer (pH 8.5) at 37°C. 1,2,3-TCP, 2,3-DCP, ECH , CPD, GDL were quantified by a gas chromatograph Fuli GC System 9750 equipped with a capillary column ZB-FFAP 30 m x 0.25 mm x 0.25 µm (Phenomenex, USA). Glycerol was quantified by a gas chromatograph Fuli GC System 9790Ⅱ equipped with DB-5 column (L60 m×i.d. 0.25 mm and df 0.25 m thin coating film) (Supelco, Bellefonate, PA, USA).

Detection of 1,2,3-TCP, 2,3-DCP, ECH, CPD, GDL

  1. Sample preparation:
    • Samples (0.25 mL) were collected from the reaction mixture, extracted with 0.5 mL of ethyl acetate and dried with anhydrous magnesium sulfate(12000×g, 90s).
  2. Gas chromatography analysis:
    • Aliquots (2 μL) were injected into the GC with an injector temperature of 220°C. The operating column temperature was 100°C. The temperature of the detector was 280°C.
    • The concentration of the products were determined by GC under the following conditions: 100 °C for 7min, 15 °C/min to 220°C, and 15 min at 220 °C.

Detection of glycerol

  1. Sample preparation:
    • One hundred microliters of the fluid sample and 50 μL of 1,2,3-butanetriol internal standard (100 μg/mL) were mixed and lyophilized. Then, the dried sample was homogeneously mixed with 200 μL of TMSIM and 50 μL of acetonitrile through ultrasonication. The mixture was placed in a 60 ◦C water bath for 45 min to perform the pre-column derivatization. The reaction solvent was then evaporated using a vacuum evaporator. The dried residue was dissolved in 100 μL of hexane.
  2. Gas chromatography analysis:
    • The GC(Fuli 9790Ⅱ) was equipped with a DB-5 column (L60 m×i.d. 0.25 mm and df 0.25 m thin coating film) (Supelco, Bellefonate, PA, USA). Nitrogen was the carrier gas. The injection port temperature was set at 280 ◦C. The oven temperature was programmed from 100 to 250 ◦C at a rate of 10 ◦C/min and was held at the final temperature for 15 min. The glycerol concentration was calculated using a calibration curve of the peak area ratios of different quantities of glycerol standard to the consistent quantity of the added internal standard.

2. Protein expression

The recombinant strain E. coli MC1061, carrying either pBADDhaA31, pBADEchA, pBADHheC-WT or pBADHheC-mutant gene, was incubated overnight at 30 °C in 500 ml LB medium containing 100 μg/ml ampicillin and 0.05% (w/v) L-arabinose to reach an OD600 of 1.7 to 2.0. Cells were harvested by centrifugation (4000×g, 15 min) at 4 °C and washed once with 50 mM Tris-SO4 buffer (pH 8.0), then centrifuge(8000×g, 10min). The concentrations of proteins were measured using Bradford’s method.

3. Enzyme activity assay

Reaction proceeded in 5 ml 50 mM Tris-SO4 buffer (pH = 8.0) at 37°C, and the reaction was initiated by the addition of a certain amount of enzyme. Samples (0.25 ml each) were taken periodically from the reaction mixture and immediately added into 0.9 ml reagent I [30 mM NH4Fe(SO4)2 in 1 M HNO3] to stop the reaction, after which 0.1 ml reagent II [saturated solution of Hg(SCN)2 in absolute ethanol] was added. The absorbance of the mixture at 460 nm was measured on a UV spectrometer.

Reagent

1. Luria-Bertani Liquid Medium (1L)

Components Contents
Typtone 10g
Yeast extract 5g
NaCl 10g

2. Luria-Bertani Solid Medium (1L)

Components Contents
Typtone 10g
Yeast extract 5g
NaCl 10g
Agar 12g

3. MSB Solid Medium (1L)

Components Contents
100x MS macroelement solution 40ml
100x MS microelement solution 10ml
100x Ferric salt solution 10ml
1000x B5 vitamin solution 10ml
Sucrose 30g
Add distilled water until the medium volume reach 1000 mL
Blend with magnetic stirrer
Adjust medium pH to 5.8 with 1 mol/L NaOH
Agar 7g

4. Antibiotics

  Antibiotic ddH2O absolute ethyl alcohol DMSO (Dimethyl sulfoxide)
1000×Kanamycin(50mg/mL) Kanamycin 1g 20ml \ \
1000×Ampicillin(100mg/mL) Ampicillin 2g 20ml \ \
1000×Chloramphenicol(30mg/mL) Chloramphenicol 0.68g \ 20ml \
500×Cefotaxime (250mg/ml) Ceftriaxone 5g 20ml \ \
1000×Rifampicin(50mg/ml) Rifampicin 1g \ \ 20ml

5. 50×TAE

Components Contents
Tris base 242g
Na2EDTA∙2H2O 37.2g
ice vinegar 57.1mL
ddH2O 800mL

6. CTAB Buffer

Components Contents Contents
CTAB 4g
NaCl 16.364g
1M Tris-HCl (PH8.0) 20ml
0.5M EDTA 8ml

7. 1 M Tris pH 8.0

Dissolve 121.1 g of Tris base in 800 ml of H 2 O. Adjust pH to 8.0 by adding 42 ml of concentrated HCL. Allow the solution to cool to room temperature before making the final adjustments to the pH. Adjust the volume to 1 L with H 2 O. Sterilize using an autoclave