LB Medium and LB Medium Solid

Our LB Medium is from Sigma-Aldrich (LB Broth - Sigma L3022-1KG), just dilute it in distilled water. Concentration: 20g/L

Materials:

• Beaker
• Graduated beaker
• Magnetic stir bar
• Distilled water
• LB medium
• Agar * (solid medium)

1. Weigh half LB in a beaker (small volume);
   For solid medium for plates, add 2% (w / v) agar;
2. Add less volume than total distilled water;
3. Place on the shaker with a magnetic stir bar until smooth;
4. Transfer to a graduate beaker;
5. Make up to volume with distilled water;
6. Return to beaker and mix on shaker until smooth again;
7. After preparation, autoclaving the medium.

Preparation of Antibiotic Stock Solutions

• Chloramphenicol (C28)
  NOTE: Chloramphenicol a light sensitive. Avoid exposing it to light!
1. Weigh mass to desired volume of solution;
   1. Beaker
   2. Falcon
2. Add volume, less than total of ethanol (95%);
3. Dissolve completely;
   1. Magnetic stir bar
   2. Vortex
4. Transfer to a graduated beaker;
5. Make up to volume with ethanol (95%);
6. Distribute 1ml for each eppendorf stock;
7. Cover with aluminum foil;
8. Store at -20ºC.
• Kanamycin (K1)
  NOTE: Poorly soluble. Attention to homogenize.
1. Weigh mass to desired volume of solution;
   1. Beaker
   2. Falcon
2. Add volume, less than total of sterile water;
3. Dissolve completely;
   1. Magnetic stir bar
   2. Vortex
4. Transfer to a graduated beaker;
5. Make up to volume with sterile water;
   (In the flow):
6. Pass solution through the syringe filter (eliminate possible microorganisms);
7. Distribute 1ml for each eppendorf stock;
8. Store at -20ºC.

Preparation of Plates with Medium and Antibiotic

Comments:

• Always work with autoclaved medium;
• Only open autoclaved medium inside the flow;
• The addition of antibiotic to make the medium selective is done only at the time of use the medium (antibiotic cannot be autoclaved, because it degrades him).
• Agar-containing medium (solid medium), after liquefying, rapidly gets hard at room temperature. Fast handling required.

1. Melt solid medium (LB + agar, autoclaved).
   1. Semi-screw cap: unscrew 1/2 turn;
   2. Microwave for 1 minute;
   3. Shake to help the liquefaction;
   4. If necessary, place for another 20' in the microwave.
      ** Only glass containers go inside the microwave (falcon tube CAN NOT go in the microwave).
      ** Keep an eye for the medium not to boil and overflow. If it begins to bubble very close to the lid, stop the microwave cycle.
2. Add antibiotic (make selective medium).
   1. Work Concentrations:
      1. Kanamycin: 50ug / ml
      2. Chloramphenicol: 35ug / ml (light sensitive: cover with Al paper)
   2. Check the total volume of medium to be worked with, to get the correct volume of antibiotic solution;
   3. Place medium (liquid), in falcons tubes, 1 for each antibiotic to add them in the medium;
   4. Wait for the moment when the tube containing the liquid medium can be held directly by hand, to add the antibiotic (temperature at which the antibiotic will not be degraded).
3. Plate the medium in petri plates.
   ** Minimum of medium per plate: 15ml.

Pre-inoculum preparation

NOTE: If the pre-inoculum is not done on the day immediately after plating, the plates with the colonies will be stored in the refrigerator at 4 ° C.

Materials:

• Autoclaved test-tubes;
• Liquid medium (selective, in case of transformed cells)
• Transfer handle

(Preparation in the flow)

(use the Bunsen burner)

(Always flambé the outlet of the glassware/handle).

1. Addition of culture medium to the test-tube (10 ml);
2. Flambé handle (wait for it to cool within the safety zone of the Bunsen burner);
3. Collect 1 transformed colony;
4. Insert the handle into the test-tube containing medium (ensure that the liquid medium has touched the point of the handle where the colony was);
5. Thread the test tube partially for aeration (½ turn);
6. Overnight growth (16h), shaking at 37°C (recommended masking tape to hold the tube cap).

DNA resuspension iGEM kit

Every plate in the kit comes with a aluminium foil protection in order to avoid cross contamination between samples. Never remove this protection. Always manipulate plates in the flow chamber:

1. Using a pipette tip, make a small hole on the well’s lid;
2. Add 10 uL of distilled water;
3. Carefully resuspend the DNA with the pipette;
4. Let the DNA rest for 5’ at room temperature for hydration;
5. DNA is ready to be used!
6. After the resuspension, store the plate at -20^oC
7. Use 1uL to perform a transformation.

Bacterial transformation 1

Manipulation in the flux and on ice:

1. Add 100 uL of competent cell in an autoclaved eppendorf (1.5 mL);
2. Add DNA (50 - 100 ng);
3. Ice 30’;
4. Thermal-shock 42ºC, 1.5’;
5. Ice 5’;
6. Add 250 uL sterile LB medium (without antibiotic);
7. Incubate for 45’, 37ºC, under agitation (150 rpm);
8. Plaque transformed cells in selective medium;
9. Incubate overnight, 37°C, static;

Miniprep - Plasmid DNA Extraction (CellCo)

NOTE: CellCo Fast-n-Easy Plasmid Mini-Prep Kit. The kit comes with all the necessary solutions/buffers, plus the column and collection tubes (fit into the column).

(Cell Lysis)

1. Add aliquot of cell culture (1 - 3 ml) in an eppendorf;
2. Centrifuge;
3. Add 300uL of 'Lysis Buffer';
4. Resuspend in the pipette for 1'.
   1. Neutralization
5. Add 300uL of 'Neutralization Buffer';
6. Mix gently by inversion, maximum of 4-6 times;
7. Centrifuge in the 10,000g microcentrifuge for 5'.
   1. Yellow color: correct pH, proceed
   2. Purple, orange color: adjust pH.

(Column Activation)

1. Add 100uL of 'Activation Buffer' to the column;
2. Place column in a 2ml collection tube;
3. Centrifuge in the 10,000g microcentrifuge for 30''.

(Column loading)

1. Add supernatant (7.), to the column;
2. Centrifuge in the 10,000g microcentrifuge for 30'';
3. Discard the eluate;
4. Place column in a 2ml collection tube;

(Column wash)

1. Add 500uL of 'Wash Buffer';
2. Centrifuge in the 10,000g microcentrifuge for 30'';
3. Discard the eluate;

   * For super purification, extra steps:

   1. Add 700uL of 'Wash Buffer';
   2. Centrifuge in the 10,000g microcentrifuge for 30'';
   3. Discard the eluate;
   4. Centrifuge in microcentrifuge 10,000g for 2';

(Elution)

1. Place column in a clean 1.5ml microtube;
2. Add 50uL of Milli-Q water;
3. Inoculate at room temperature for 1';
4. Centrifuge in the 10,000g microcentrifuge for 1' to elute the DNA.

Oligos resuspension (EXXTEND)

Preparation of Resuspension Solution

First, prepare Tris 1M and EDTA 0.5M, according to the following protocols:

• 1M TRIS (Tris (hydroxymethyl) aminomethane) (pH = 8.0) (Volume = 1L):
  • Base Tris: 121.12g;
  • Adjust pH 8.0 with HCl;
  • Complete the sufficient amount for 1000 ml with Milli-Q H2O.

• EDTA (Ethylenediaminetetraacetic Acid) 0.5M (pH = 8.0) (Volume = 50ml):
  • EDTA: 9.305g (MW = 372.22);
  • Adjust pH = 8.0 with concentrated NaOH;
  • Complete the sufficient amount for 50 ml with Milli-Q H2O.

Autoclave EDTA and Tris solutions before preparing TE 1X buffer!

• 1X TE Buffer Solution [10 mM Tris-HCl, 0.1 mM EDTA (pH 7.5 - 8.0)] (Volume = 500ml)
  • Add 5 ml of 1M Tris;
  • Add 0.1 ml of 0.5M EDTA;
  • Add 494.9 ml of Milli-Q H2O.

Store 1X TE at room temperature in a closed bottle with a lid and parafilm.

Resuspension of the oligos

Exxtend synthesized oligos are shipped dry as they are highly stable in this form.

(After receiving the oligo):

1. Centrifuge the tube to make sure that any dry oligo that has come off the tube bottom during shipment is returned to it;
2. To resuspend the oligo use TE buffer solution (10 mM Tris, 0.1 mM EDTA, pH 8.0);
3. Let the oligo hydrate for a few minutes with intermittent vortex before quantification.

** It is important to note that dilution of the stock solution to working concentration should always be done in pure DNA free water.

Preparation of 100 µM oligo stock solution

Exxtend provides an oligo specification sheet stating quantitative data in nmoles, optical density (OD) units, micrograms (µg) and molecular weight (PM). This allows you to calculate the stock solution at any concentration value you prefer. The general rule is that for any oligo, the number of nmoles x 10, will give you the amount of solvent, in microliters, to obtain 100 µM solution (pmol / µL).

[Molar Concentration] x [Volume] = [mol number], where [1 nmol = 1000 pmol]

Therefore,

[Volume in µL] = [nmol] x [1000 pmol / 100 µM (pmol / µL)] = nmol x 10

The oligo specification sheet also shows the dilution volume in µL for 100 pmol/µL (µM) concentration. Using this volume of solution, your oligo will be suspended at the stock concentration (100 pmol/µL = 100 µM).

Remember that the oligos should be diluted in TE solution (10 mM Tris, 0.1 mM EDTA, pH 8.0), and diluted to working concentration (5-10 µM) in pure water free of DNAses.

Synthetic DNA Resuspension (Twist Bioscience)

NOTE: Twist Bioscience products are dried and shipped in 2ml microcentrifuge tubes or 96-well plates. Although double stranded DNA and single stranded oligonucleotides are stable under most standard laboratory storage conditions, it is important to consider the following best practices to maintain the high quality of Twist Bioscience synthesized DNA.

1. Upon receipt, centrifuge the tube or plate briefly and resuspend in nuclease-free Tris-EDTA (TE) buffer, pH 8.0 or 10 mM Tris-HCl, pH 8.0 to the desired concentration. We do not recommend resuspension in water.
2. A concentration of at least 10 ng/uL is recommended for stock dilution, but the optimal concentration will need to be determined based on the application of interest.
3. Prepare aliquots of stock dilution solution and separate working aliquots to limit the chance of contamination and reduce the number of freeze/thaw cycles. Use working aliquots as soon as possible after preparation and minimize exposure to high temperatures.
4. For long term storage (over 1 year), freeze the DNA at -20 °C. For even longer storage, freeze the DNA at -80 °C.

Preparation 0.8% agarose gel

NOTE: Due to the use of ethidium bromide in the agarose gel, the entire gel preparation step should be performed in the BrEt specific laboratory area. Always handle nitrile gloves. Do not leave the specific area with the gloves employed in the process.

(In the BrEt area):

1. Prepare the gel base on the stand by leveling it;
2. Choose the comb with the desired amount/size of wells and fit into the base of the gel;
3. Weigh agarose in the conical flask;
4. Add 1X TAE buffer volume;
   1. If missing, dilute TAE 25X Buffer: 24 distilled water to 1 TAE 25X;
5. Solubilize agarose, use of microwaves;
   1. 30 seconds in the microwave;
   2. Shake erlenmeyer;

      * Hot glassware, handle with the help of a paper towel;

      1. If visible agarose particulate matter, repeat microwave oven every 10 seconds, being careful not to boil the mixture;
      2. Cool the mixture under tap water by shaking the conical flask until it can be held by hand;

         * Do not overcool to prevent early solidification.

         1. Add ethidium bromide;
         2. Stir to homogenize;
         3. Pour the mixture into the base of the gel;
            1. If there are bubbles, with a tip, take them to the end of the gel removing them from the race path.
         4. Wait for gel to harden until opaque (10~15 minutes);
         5. Put the base in the tub

            * Place the gel with the wells side to the same side of the black electrode.

            1. Fill the bowl with TAE 1X to the maximum height mark;
               1. Ensure that the gel is completely covered.
            2. Connect the cover wires to the source (black to black and red to red);
            3. Turn on the source (switch);
            4. Set the desired parameters;
            5. Put the lid on the bowl;
            6. Press the ON/OFF button to start the race;
               1. If all is correct, small bubbles will rise on both sides of the tub.
            7. After the race is over, press the ON/OFF button to stop and remove the cover.

Purification of PCR product from agarose gel

NOTE: Promega Kit 'Wizard SV Gel and PCR Clean-Up System'. The kit comes with all the necessary solutions, besides the column and collection tube (fits the column).

NOTE 2: Due to the use of ethidium bromide in the agarose gel, the entire gel manipulation step should be performed in the BrEt specific laboratory area.

(Agarose Gel Dissolution)

(Specific Area BrEt)

1. Extract gel band with corresponding DNA (cut with blade in UV transilluminator);

   * Minimum possible UV time (avoid sample degradation).

   1. Weigh gel slice:
      1. Tare the eppendorf of 1.5 mL;
      2. Add gel slice to eppendorf;
      3. Obtaining the extracted gel mass.
   2. Add 10 μL of Membrane Binding Solution to each 10 mg of gel;
   3. Vortexing;
   4. Incubate at 50-60 °C until the gel has completely dissolved;

(DNA binding to column)

1. Place a mini column in a collection tube;
2. Add all volume of gel to the column and incubate at room temperature for 1 minute;
3. Centrifuge at 16000 g or 14000 rpm for 1 minute;
4. Remove column from collection tube and discard passing liquid;
5. Return the column to the collection tube;

(Column wash)

1. Add 700 µL of 'Membrane Wash Solution';
2. Centrifuge at 16000 g or 14000 rpm for 1 minute;
3. Discard the passed liquid;
4. Add 500 µL of 'Membrane Wash Solution';
5. Centrifuge at 16000 g or 14000 rpm for 5 minutes;
6. Discard the passed liquid;
7. Centrifuge with the centrifuge lid open for 1 minute, for ethanol evaporation (IMPORTANT);

(DNA Elution)

1. Transfer the mini column to a clean 1.5 mL eppendorf;
2. Place 50 µL of free-water nuclease (heated to 60 °C), directly in the center of the column but not touching the membrane.

   * Do not use the nuclease free-water kit.

   1. Incubate at room temperature for 1 minute;
   2. Centrifuge for 1 minute at 14000 rpm;
   3. Discard mini column and store DNA at 4 °C or -20 °C.

Calcium chloride competent cells

CaCl2 Solution - pH 7.0

(Day 1 - Preparations):

1. Streaking in LB the cell of interest (DH5ɑ / BL21);
2. Autoclaving 100 ml of liquid LB;
3. Autoclaving 100 ml CaCl 2;
4. Autoclaving 2 centrifuge tubes of 250 ml;

(Day 2 - Preparations):

1. Make a 5 ml post-inoculum with a plate colony and leave overnight at 37 °C and 250 rpm;

(Day 3 - Competent Cells):

(On ice! Work with everything cold, including the solution)

1. Place 2 ml pre-inoculum in the 100 ml medium and incubate at 37 °C and 250 rpm until OD = 0,375;
2. Distribute the culture in 2 centrifuge tubes (cold), and centrifuge at 1600g for 7min and at 4ºC;
3. Resuspend the pellet in 10 ml of CaCl2 solution, shake with circular movements in the hand (cells are fragile);
4. Resuspend the pellet in 10 ml of CaCl2 solution and incubate for 30 min on ice;
5. Centrifuge again at 1100g for 5min and at 4 °C;
6. Resuspend the precipitate in 1 ml of cold CaCl2 solution;
7. Transfer to a sterile 1.5 ml eppendorf;
8. Aliquot 50ul;
9. Freeze in liquid nitrogen;
10. Store at -80ºC.

PCR - Q5 High-Fidelity 2X Master Mix (NEB)

The protocol used for this PCR can be found in the official NEB website (https://international.neb.com/), and it is this one:

https://www.neb.com/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492

Biofilm quantification using violet crystal

A three days protocol.

• Day 1: Preparations
  1. Pre inoculum (5mL LB medium + antibiotic), overnight growth, 37ºC, under agitation;
  2. Medium SOC liquid, including erlenmeyers (125 mL) with 20 mL for each construction;
  3. Violet crystal 0,1% solution;
  4. Acetic acid 30% solution;
  5. PBS buffer;
  6. Autoclaved water;
  7. Sterile 24-well plate;
  8. Sterile (or not) 96-well flat bottom plate.
• Day 2: Experiment set up
  1. Pre inoculum’s OD measurement (600nm)
     1. Dilution 1:1 in LB medium for measurement in spectrophotometer
  2. Calculations for culture’s inicial OD (60nm) = 0.1, in the final volume of 20 mL of SOC medium (erlenmeyer 125 mL):
     ODincitial ✕ V mL = 0.1 ✕ 20 mL
     V: pre inoculum’s volume
  3. Inoculation of SOC medium prepared in erlenmeyer (125 mL), shaker 150 rpm, 37ºC, about 4 hours for growth rate conference;
     Biofilm production
  4. Adding cells and SOC medium + antibiotic in the plate, so that the culture starts it’s biofilm production with 2 mL total volume, OD (600nm) = 0.2;
     1. Calculation of the inoculum volume:
        ODinitial ✕ V mL = 0.2 ✕ 2 mL
        V: inoculum’s volume
     2. Complete to 2 mL with SOC medium + atb
  5. Example of 24-well plate assembly:

     Blank	Control	Control	Control	Control	Blank
     Blank	Samples A	Samples A	Samples A	Samples A	Blank
     Blank	Samples B	Samples B	Samples B	Samples B	Blank
     Blank	Samples C	Samples C	Samples C	Samples C	Blank

     1. Blank: 2 mL SOC + atb
     2. Control: Culture without the plasmid in SOC medium + atb
     3. Samples: V(DO600 = 0.2) Culture + V (to 2 mL) SOC medium + atb
        ** In case of lac operator, add 2 uL IPTG (1M)
  6. lncubate for 24 hours, 37ºC, under agitation (150 - 180 rpm);
  7. After 24 hours, begin the biofilm quantification.
• Day 3: Biofilm quantification
  1. Pour the plate in a discard reservatory with sodium hypochlorite;
  2. Wash gently with PBS 3 times;
  3. Let it dry at room temperature. The excess can be removed with absorbent paper and then let the plate dry face up;
  4. Add 2 mL methanol (in fume hood), close the plate and 15 minutes in refrigerator (~15ºC);
  5. Pour the methanol (in fume hood) in an proper discard;
  6. Press the plate against an absorbent paper and let it dry face up to evaporation of the fixer;
  7. Add 2 mL violet crystal, wait 20 minutes;
  8. Pour the violet crystal and use water to wash gently the plate;
  9. Remove water excess with an absorbent paper;
  10. Add 2 mL acetic acid 30% and homogenize each well;
  11. Transfer 125 uL of each well to a 96-well flat bottom plate for Abs reading in 590nm.

Preparation mercury (Hg) solutions

Attention: mercury is toxic. Working with it requires safety measurements: EPI’s.
Solutions of 1ml in 3 concentrations: 20 mg/mL, 200 ug/uL and 20ug/uL of the mercury salt mercury chloride (HgCl2).

• Solution [HgCl2] = 20 mg/mL
  1. Weight the mass of HgCl2: 20 mg;
  2. Add 1 mL water (Milli-Q);
• Solution [HgCl2] = 200 ug/mL
  1. Dilute the 20 mg/mL solution: in a eppendorf (1.5 mL) add 10 uL of 20 mg/uL solution and complete with 990 uL of water (Milli-Q);
• Solution [HgCl2] = 20 ug/mL
  1. Dilute de 200 ug/mL solution: in a eppendorf (1.5 mL) add 100 uL of 200 ug/uL solution and complete with 900 uL of water (Milli-Q);

Hg Disk Diffusion Test

Obs: protocol developed by the team in order to confirm that transformed bacteria are indeed able to survive in Hg contaminated media.

Materials:

• paper filter cut in circles of ~10mm diameter
• Hg salt (HgCl₂)
• MilliQ water
• Pre-inoculated transformed bacteria
• Petri dishes

Methods:

1. Set pipette for 10 uL to soak paper filter disc in Hg solution
2. While the disc dries, spread pre-inoculated cells over prepared Petri dish
3. Using a sterilized tweezers, pinch Hg dripped disc
4. Carefully place it over the agar medium
5. Close plate and let it incubating for the experiment time span of interest.

Quantification of coconut fiber biofilm

Materials:

• PBS;
• methanol;
• 30% acetic acid;
• Violet crystal 0.1%;
• Plate with 24 wells (fiber);
• Flat plate with 96 bottom wells.

(Qualitative visual analysis: photos):

(Quantification):

1. Fiber-board separation:
   1. Remove the fiber from each well and place it in a new plate with 24 wells;
   2. Pour the plate into an ice cream pot (with hypochlorite - bleach).
2. Wash gently with 3X PBS buffer;
   1. ** For fibers, remove one by one and position in the respective location of the well on the lid. Wash the plate and then the fiber one by one, returning them to their respective wells.
3. Allow to dry at room temperature;
4. Add 2ml methanol (chapel), (total 32ml / plate). Put the lid and than in the refrigerator for 15 min;
5. Pour methanol (organic solvents), into a pot and discard properly.
   1. ** For fibers, remove one by one and position in the respective location of the well of the lid. Pour the plate and return them;

      (Work in the chapel)

      1. Take the amount in a beaker (100ml);
      2. Discard in an ice cream jar;
      3. In the end, discard everything, including the unused, in the discard pot.
   2. Squeeze the plate against a paper and let it face up;
   3. Add 2ml violet crystal and leave for 20 min;
   4. Pour the violet crystal and gently wash the plate with milli-Q water;
      1. ** For fibers, remove one by one and position in the respective location of the well on the lid. Wash the plate and then the fiber one by one, returning them to their respective wells.
   5. Remove excess water with absorbent paper;
   6. Add 2ml of 30% acetic acid (total 32ml / plate), and homogenize each well;
   7. Transfer 125ul from each well to a flat plate with 96 bottom wells, for the Abs reading at 590nm.

Coconut fiber production

NOTE: We bought the green coconut in the market.

Materials:

• Green coconut
• Knife
• Beakers
• Blender
• Sieve
• Distilled water
• Use the knife to open a window in the coconut.
• Remove the coconut water.
• Cut the coconut into some slices using the knife.
• Remove epicarp, endocarp and edible part.
• Insert pieces of the mesocarp of about 5g in the blender and add distilled water until it covers them.
• Beat in blender for about 2 min.
• Remove from the blender and, using a sieve, wash the fiber with distilled water.
• Place the washed fiber in beakers and allow it to dry in a humid incubator at 37ºC, for about 8h.