Team:ITB Indonesia/Experiments

Protocols

Materials :
  • Escherichia coli TOP10 Culture
  • LB agar plate
  • Super Optimum Broth (SOB)
  • CCMB80 buffer (from: http://parts.igem.org/Help:Protocols/Competent_Cells)
  • SOB (from : http://parts.igem.org/Help:Protocols/SOB)
  • Ice bucket
  • 50 mL and 15 mL Falcon
  • 100 mL Erlenmeyer Flask
  • 1.5 microcentrifuge tube
Protocol :
  1. Streak E.coli from glycerol stock to LB plate
  2. Incubate the plate overnight (16-18 hrs)
  3. Inoculate 5 mL SOB in 15 mL sterile Falcon with E.coli from the plate, then incubate it overnight in 37oC with continuous shaking at 200 rpm
  4. Inoculate 47.5 mL SOB in 100 mL sterile Erlenmeyer with 2.5 mL overnight E.coli. Incubate it in 37oC with continuous shaking at 200 rpm until it reach OD600 0.3-0.4 (usually 20-30 minutes for TOP10)
  5. Aliquot @15 mL culture to 15 mL sterile Falcon, then centrifuge it at 3000g, 4oC, for 10 minutes. Discard the supernatant
  6. Resuspend the cell pellet with 8 mL ice cold CCMB80 buffer and incubate it on ice for 20 minutes
  7. Centrifuge the resuspended culture at 3000 g, 4oC, for 10 minutes. Discard the supernantant
  8. Resuspend the pellet with 1 mL Ice cold CCMB80 buffer and incubate it for 20 minutes
  9. Aliquot the chemically competent cell @50 µL to 1.5 mL microcentrifuge tube and store it in -80oC until usage
Safety Considerations :
  • Good Microbiological Practice (GMP) is necessary (e.g using protective and safety equipment, using aseptic technique, minimizing possibility of producing aerosols)
  • After work, all the materials that exposed to cell culture should be autoclaved
Materials :
  • Escherichia coli TOP10 chemically competent cells
  • LB agar plate with appropriate antibiotic
  • SOC (SOB with 20 mM glucose,from : http://parts.igem.org/SOC)
  • 1.5 mL microsentrifuge tube
Protocol :
  1. Chill the chemically competent E.colicoli TOP10 culture (in 50 µL aliquot) in ice bucket for 10 minutes
  2. Add 1-3 µL plasmid to the aliquot and incubate it on ice bucket for 30 minutes
  3. Incubate it on waterbath with 42oC temperature for 1 minute
  4. Immediately move the tube to ice bucket and incubate for 5 minutes
  5. Add 200 µL SOC to the tube and incubate it for 2 hours in 37oC, 200 rpm
  6. Innoculate the transformed culture @100 µL to LB plate with appropriate antibiotic, with spread plate method for easier single colony pick
  7. Incubate the inoculated plate overnight in 37oC
Safety Considerations :
  • Good Microbiological Practice (GMP) is necessary (e.g using protective and safety equipment, using aseptic technique, minimizing possibility of producing aerosols)
  • After work, all the materials that exposed to cell culture should be autoclaved
Materials :
  • Dream Taq Green DNA Master Mix (2x) (Thermo Scientific)
  • Nuclease Free Water
  • Appropriate primer pair
  • PCR tube
  • Filtered tips
  • Ice block
Protocol :
  1. For Colony PCR, prepare sample by dilluting picked colony in 50 µL NFW
  2. On ice, add PCR components (Dream Taq DNA polymerase, primer, NFW, and template) to make 10 µL reaction
    3. Components Volume (µL)
    Dream Taq Green DNA Master Mix (2X) 5 µL
    F primer 1 µL
    R primer 1 µL
    NFW 1 µL
    Template 2 µL
  3. Vortex mix and spin down all reactions
  4. Put all samples in Thermal cycler and set the reactions as the table below
    5. Reaction Temperature (oC) Time Number of Cycle(s)
    Initial denaturation 95 3 min (colony: 4 min) 1
    Denaturation 95 30 seconds 25-40
    Annealing Appropriate temp 30 seconds
    Elongation 72 1 min/lb
    Final Extension 72 5-15 min 1
    Storage 4 - -
Safety Considerations :
  • Good Microbiological Practice (GMP) is necessary (e.g using protective and safety equipment, using aseptic technique, minimizing possibility of producing aerosols)
  • Filtered tips used for PCR preparation and performed in laminar air flow cabinet to prevent contamination in reagen
  • After work, all the materials that exposed to cell culture should be autoclaved
Materials :
  • T4 DNA ligase (NEB)
  • T4 DNA ligase buffer (NEB)
  • 1.5 mL microtubes
Protocol :
Ligation reactions were done following NEB protocol (from : https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202)
  1. To make 20 µL reactions, mix 2µL T4 DNA ligase with 1:3 molar ration of vector to insert, NFW to 20µL, and 1µL T4 DNA ligase. Mixing performed with the tube placed on ice.
  2. Mix all reagents by pipetting up and down, continued with spin down
  3. Incubate reactions in 37oC overnight
  4. Heat inactivate at 65oC for 10 minutes and chill the reactions on ice for transformation into competent cells
Safety Considerations :
  • Wearing personal protective safety equipment is necessary
  • Keep working area clean and tidy
  • Follow necessary handling and disposal considerations by the manufacturer
This protocol was performed to check the restriction reactions, PCR and plasmid isolation.
Materials :
  • Agarose powder
  • TAE buffer 1X (Tris, Acetate, EDTA)
  • Loading dye 6X
  • DNA ladder 1Kb
  • EtBr (Ethidium Bromide)
  • ddH2O
Protocol :
  1. Dissolve 1g in 100 mL TAE 1X for 1% agarose and heat the mixture in microwave for 1-3 minutes or until the mixture looked clear
  2. Pour agar mixture in the gel cast and place a well comb. Wait until the gel solidify
  3. Place gel in electrophoresis chamber filled with 1X TAE buffer
  4. Insert mixture of DNA ladder and loading dye, and insert all samples (add loading dye if the sample doesn’t include one)
  5. Run electrophoresis at 70V, for about 30-40 minutes
  6. Visualize the DNA by staining the gel with EtBr solution, and destain it before observation
  7. Place the gel on UV transilluminator
Safety Considerations :
  • Wear personal protective safety equipment for performing this protocol
  • EtBr is a toxic mutagen, use double protection e.g double gloves
  • Used agarose gel disposed as cytotoxic waste
Media :
We used Luria bertani media for all E.coli cultivation.
Materials :
  • Tryptone (1%)
  • Yeast Extract (0.5%)
  • NaCl (1%)
  • For Solid media, Agar A (2%)
  • Distilled water
Media preparation :
To make LB media, mix all ingredients and dissolved them in distilled water. We usually pre heat the agar media solution in hotplate.
All LB media must be sterilized by autoclave.
For E.coli culture in solid media :
We usually inoculate E.coli in solid media using two methods namely streak and spread plate. Streak method used for rejuvenating culture, while spread plate used for spread the culture evenly in the agar plate so the single colonies are formed.
Spread plate: 100µL culture inoculated to the surface of solid LB media then spreaded using sterilized spreader
Streak: colonies are picked using sterile inoculating loop and gently scratched to agar plate
For E.coli culture in liquid media :
If the inoculum is liquid, we used micropipette equipped with sterilized tips to transfer the inoculum into fresh media. If the inoculum is grown in the solid media, we picked the colonies using inoculating loop and dissolve them in the fresh media. We used 200 rpm and 37oC as a default condition for growing E.coli.
Safety Considerations :
  • Good Microbiological Practice (GMP) is necessary (e.g using protective and safety equipment, using aseptic technique, minimizing possibility of producing aerosols)
  • After work, all the materials that exposed to cell culture should be autoclaved
To convert OD600 measurement into cell number, the standard curve is needed.
Materials :
  • V.parahaemolyticus culture
  • Sea water LB broth media (minus NaCl, dissolved in sea water)
  • Sea water LB solid media
  • Sea water
Protocol :
  1. V.parahaemolyticus on solid media are inoculated to sea water LB broth media using inoculating loop. Culture are then incubated 24 hours at room temperature (RT) with 150 rpm agitation
  2. 24 hrs V.parahaemolyticus culture are inoculated to fresh sea water LB broth (10%) and incubated for 24 hrs again at RT, 150 rpm.
  3. 10% culture are inoculated to fresh sea water LB broth and incubated for 12 hrs at RT,150 rpm.
  4. After 12 hrs, aliquot the culture to some falcon tubes and set the OD600 on each falcon different by diluting them in fresh sea water LB broth. Aim for OD600 0.2, 0.4, 0.5, 0.6 and 0.7.
    Perform serial dilution to each cell culture in the falcon with 10X dilution on each transfer. Culture with dilution factor 10-4, 10-5, and 10-6 are inoculated to sea water LB solid media using spread plate method
  5. Incubate the plate in 37oC for 24-48 hrs and count the colonies that appear on the plate in the range of 30-300, multiply with 10 and dilution factor.
  6. Plot the OD600 to cell numbers
Safety Considerations :
  • Good Microbiological Practice (GMP) is necessary (e.g using protective and safety equipment, using aseptic technique, minimizing possibility of producing aerosols)
  • After work, all the materials that exposed to cell culture should be autoclaved
To get the information of V.parahaemolyticus growth in the flask, we make its growth curve by measuring its OD600¬ every 2 hours for 14 hours.
Materials :
  • V.parahaemolyticus culture
  • Sea water LB broth media (minus NaCl, dissolved in sea water)
  • Erlenmeyer Flask
Protocol :
  1. Inoculate V.parahaemolyticus on solid media to sea water LB broth media using inoculating loop. Incubate the culture for 24 hours at room temperature (RT) with 150 rpm agitation
  2. 24 hrs V.parahaemolyticus culture are inoculated to fresh sea water LB broth (10%) and incubated for 24 hrs again at RT, 150 rpm.
  3. Inoculate 10% culture to fresh sea water LB broth and incubate for 12 hrs at RT,150 rpm.
  4. Inoculate 10% culture to fresh sea water LB broth and incubate at RT,150 rpm for 14 hrs with OD600 measurement every 2 hrs using spectrophotometer UV-Vis.
  5. Convert the OD600 data to cell numbers and plot the growth over time
Safety Considerations :
  • Good Microbiological Practice (GMP) is necessary (e.g using protective and safety equipment, using aseptic technique, minimizing possibility of producing aerosols)
  • After work, all the materials that exposed to cell culture should be autoclaved
Plasmid DNA Isolation was carried out using the commercial kit from Geneaid Biotech Ltd. with the following instructions explained below.
Materials :
Equipment :
  • Microcentrifuge
  • Pipettes (1000 µL and 100 µL)
Consumables :
  • PrestoTM Mini Plasmid Kit
  • 1.5 mL microcentrifuge tubes
  • Pipette tips (1000 µL and 100 µL)
Protocol :
  1. Use 1.5-7.0 ml of cultured bacterial cells, pellet cells in a microcentrifuge for 1 minute at 14,000-16,000 g. Discard the supernatant.
  2. Add 200 µL of PD1 Buffer (make sure RNAse A was added). Resuspend the cell pellet completely by pipetting.
  3. Add 200 µL of PD2 Buffer. Resuspend sample then mix gently by inverting the microcentrifuge tube 10 times. Let stand at room temperature for at least 2 minutes to ensure the lysate is homogenous. Do not exceed 5 minutes
  4. Add 300 µL of PD3 Buffer then mix immediately by inverting the tube 10 times. Centrifuge at 16,000 g for 8 minutes. During centrifugation, place a PDH Column in a 2 mL Collection Tube.
  5. Transfer all of the supernatant to the PDH Column. Centrifuge at 14,000-16,000 g for 30 seconds, discard flow-through.
  6. Add 600 µL of Wash Buffer into the PDH Column. Centrifuge 14,000-16,000 g for 30 seconds. Discard the flow through then place the PDH Column back in the collection tube. Centrifuge 14,000-16,000 g for 3 minutes to dry the column matrix. Transfer the dried PDH Column to a new microcentrifuge tube.
  7. Add 50 µL of Elution Buffer into the center of the column matrix. Let stand for at least 2 minutes to allow elution buffer completely absorbed. Centrifuge 14,000-16,000 g for 2 minutes.
Safety Considerations :
  • Good Microbiological Practice (GMP) is necessary (e.g using protective and safety equipment, using aseptic technique, minimizing possibility of producing aerosols)
  • After work, all the materials that exposed to cell culture should be autoclaved
Gel purification was carried out using the commercial kit from Geneaid Biotech Ltd. with the following instructions explained below.
Materials :
Equipment
  • Microcentrifuge
  • Pipettes (1000 µL and 100 µL)
  • Scalpel
Consumables
  • PrestoTM Mini Plasmid Kit
  • 1.5 mL microcentrifuge tubes
  • Pipette tips (1000 µL and 100 µL)
Protocol :
  1. Cut the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice. Transfer up to 300 mg of the gel slice to a 1.5 mL microcentrifuge tube.
  2. Add 500 µLof Gel/PCR Buffer to the sample then mix by vortex.
  3. Incubate at 55-60oC for 10-15 minutes or until the gel slice is completely dissolved.
  4. Place DFH Column in a 2 mL Collection Tube.
  5. Transfer 800 µL of the sample mixture to the DFH Column.
  6. Centrifuge at 14.000-16.000 g for 30 seconds.
  7. Discard the flow-through then place the DFH Column back in the 2 mL Collection Tube.
  8. Add 400 µL of W1 Buffer into the DFH Column.
  9. Centrifuge at 14.000-16.000 g for 30 seconds then discard the flow-through.
  10. Place the DFH Column back in the 2 mL Collection Tube.
  11. Add 600 µL of Wash Buffer into the DFH Column and let stand for 1 minute. Centrifuge at 14.000-16.000 g for 30 seconds then discard the flow-through.
  12. Place DFH Column back in the 2 mL Collection Tube.
  13. Centrifuge at 14.000-16.000 g for 3 minutes to dry the column matrix.
  14. Transfer the dried DFH Column to a new 1.5 microcentrifuge tube. Add 50 µL of Elution Buffer into the center of the column matrix. Let stand for 2 minutes.
  15. Centrifuge at 14.000-16.000 g for 2 minutes.
Safety Considerations :
  • Wearing personal protective safety equipment is necessary
  • Keep working area clean and tidy
  • Follow necessary handling and disposal considerations by the manufacturer

Restriction BsaI

Restriction enzyme digestions were performed for restricting BsaI recognition site.
Materials :
Equipment :
  • Pipette
  • Cool Box
  • Incubator (37oC)
  • Heat Block
Consumables :
  • Nuclease-free water
  • CutSmart Reaction Buffer (10X) – NEB
  • BsaI
  • DNA Sample
Protocol :
  • The reaction components must be taken in a cool box filled with ice, added the components in order (water, buffer, DNA and enzymes) to a microcentrifuge tube.
    • Component 50 µL Reaction
    DNA 1 µg
    10X CutSmart Buffer 5 µL (1X)
    BsaI 1.0 µL (or 10 units)
    Nuclease-free water to 50 µL
  • Mix gently and incubate 1 hour at 37oC.

Double Digest EcoRI dan XbaI

Restriction enzyme digestions were performed for restricting EcoRI and XbaI recognition site.
Materials :
Equipment :
  • Pipette
  • Cool Box
  • Incubator (37oC)
  • Heat Block
Consumables :
  • Nuclease-free water
  • CutSmart Reaction Buffer (10X) – NEB
  • BsaI
  • DNA Sample
Protocol :
Digestion reactions were carried out following the NEB Protocol.
  1. The reaction components must be taken in a cool box filled with ice, added the components in order (water, buffer, DNA and enzymes) to a microcentrifuge tube.
    2. Component 25 µL Reaction
    DNA 1 µg
    2X TangoTM Buffer 5 µL
    EcoRI 1.0 µL (or 10 units)
    XbaI 1.0 µL
    Nuclease-free water to 25 µL
  2. Mix gently and incubate 1 hour at 37oC.
Safety Considerations :
  • Wearing personal protective safety equipment is necessary
  • Keep working area clean and tidy
  • Follow necessary handling and disposal considerations by the manufacturer

Parts Characterization

Materials :
  • LB broth media, LB broth with 25, 35, and 45 ppt salinity and 0, 10, 20 mM nitrate (Potassium nitrate was used) (supplemented with chloramphenicol)
  • Plasmid-bearing E.coli TOP10 (plasmid containing BBa_K381001 part)
  • 15 mL centrifuge tubes
Protocol :
  1. Inoculate Plasmid-bearing E.coli TOP10 from solid media to normal LB broth, LB broth with 25,35, and 45 ppt salinity with variative nitrate concentrations (1 oose loop of bacteria to 7 mL broth) and incubate the culture overnight at 37oC, 200 rpm.
  2. After being incubated overnight, check the fluorescence with Glomax by inoculating 200 µL culture to 96 well plate and run a fluorescence reading.
Safety Considerations :
  • Good Microbiological Practice (GMP) is necessary (e.g using protective and safety equipment, using aseptic technique, minimizing possibility of producing aerosols)
  • After work, all the materials that exposed to cell culture should be autoclaved
Materials :
  • LB broth media (supplemented with chloramphenicol and ampicillin)
  • Sea water LB media
  • Wild-type Vibrio parahaemolyticus
  • Plasmid-bearing E.coli TOP10 (plasmid containing BBa_K3252018 part)
  • 50 mL centrifuge tubes
Protocol :
  1. Inoculate Plasmid-bearing E.coli TOP10 from solid media to normal LB broth that has been added with ampicillin and cloramphenicol (3 oose loops of bacteria to 15 mL broth) and incubate the culture overnight at 37oC, 200 rpm.
  2. At the same time, inoculate wild-type Vibrio parahaemolyticus from solid media to sea water LB media and incubate the culture overnight at room temperature, 150 rpm.
  3. After being incubated overnight, centrifuge V. parahaemolyticus’ culture to gain its supernatant, and filter the supernatant with 0.22 um membrane filter.
  4. Mix the newly harvested supernatant to fresh LB broth media containing ampicillin and chloramphenicol, and make variations of 0%, 10%, 50% supernatant-mixed LB broth media.
  5. Inoculate overnight culture of plasmids-bearing E. coli TOP10 to the newly made supernatant-mixed LB broth media (150 uL culture to 15 mL media).
  6. Incubate inoculated culture at 37oC, 200 rpm.
  7. Check fluorescence and OD600 of incubated culture every hour by transferring 100 uL of each culture to transparent 96-well plate and run fluorescence and absorbance reading with Glomax Plate Reader by Omega.
Safety Considerations :
  • Good Microbiological Practice (GMP) is necessary (e.g using protective and safety equipment, using aseptic technique, minimizing possibility of producing aerosols)
  • After work, all the materials that exposed to cell culture should be autoclaved
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