Synthetic biology protocols

Chemocompetent bacteria:

1. Inoculate 500 ml LB with 5 ml of the overnight culture and incubate it shaking at 37 ° C till the OD = 0.4.
2. Cool the culture for 10 min. in some ice.
3. Centifruge the sample at 6000 rpm at 4 °for 3 minutes.
4. Suspend the precipitate gently in ~ 20 ml of cold solution of 0.1 M CaCl2, then add 0.1 M CaCl2 to a volume of 300 ml.
5. Centrifuge again as above.
6. Suspend the precipitate in 10 ml of cold 0.1 M CaCl2 and incubate it for 30 min.
7. Centrifuge again.
8. Suspend the sample in 6 ml of 0.1 M CaCl2 and 15% glycerol and Pipette 50 ul to an eppendorf (put them immediately in liquid nitrogen) and store at -80 °C.

Transformation of chemocompetent bacteria:

1. Put the bacteria into ice for 2-3 minutes (or until it melts)
2. Add a cooled plasmid or ligation in a volume not bigger than 20 ɰl and stir with a tip
3. Keep it in the ice for 20 to30 minutes
4. Put it to a heating block set for 42°C for 1.5 min
5. Put it back to ice for 2 min
6. Add 900 ɰl of SOB and incubate at 37°C for 1 h
7. Sow on the appropriate selective deposit.

Transformation of electrocompetent bacteria

1. Put the bacteria into some ice for 2-3 minutes (or until it melts).
2. 50 ɰl of bacteria pipete to a dialised and cooled plasmid DNA or ligation
3. Put the transformation mixture to a cuvette (also previously cooled on ice) - it is important that the sample must be on the bottom of the cuvette and free of air bubbles. You can tap the cuvette several times on the table.
4. Transform the bacteria in an electroporator set on 2500 V (time constant should be around 5)
5. Immediately add 900 ɰl of SOB and incubate at 37°C for 1 h
6. Sow the appropriate selective deposit

Cellular biology protocols

MTT assay

In order to measure cytotoxity of acrylamide we decided to implement MTT assay. It bases on the conversion of the tetrazolium dye- MTT to insoluable formazan by cellular enzymes. Because formazan gives a purple colour, we are enabled to measure it’s intensity at 570nm using the spectrophotometer. Cellular metabolic activity reflects the number of viable cells present in the probe so we expect a loss of purple colouring in the wells where acrylamide was added.

Protocol:

1st Day:

• Removing media
• Trypsinizing and counting cells
• Adding approximately 40 000 cells with fresh media into each well of 12-well plate

2nd Day:

• Treating cells with acrylamide: adding definite volume of stock solution of acrylamide to each well in order to obtain demanded concentration
• Adding DMSO to the control

How to find the proper amount of acrylamide?

• demanded concentration x volume (1 ml for the 12-well plate) = 10³ x sought volume

3rd Day:

• examining with pipette how much liquid remains in each well
• removing excess media, leaving only 300 µl in each well
• adding 2 times less MTT- 150 µl
• incubating in 37°C for 2 hours
• removing media with an aspirator
• adding DMSO 100-500 µl transfering from each well of 12-well plate 100 µl of dissolved formazan on each well of 96-well plate. Using a few (3-5) wells of 96-well plate for each well of 12-well plate is recommended
• reading absorbance at 570 nm

Hoechst stain

Hoechst 33342 stain protocol

In order to be able to examine cell’s nuclei morphology we introduced staining with Hoechst dye. The dye binds to the minor groove of dsDNA preferentially to sequences rich in adenine and thymine. As the Hoechst dye may be excited by ultraviolet light at around 350 nm, and then emits blue fluorescent light (maximum at around 461 nm) we’ll be able to observe changes in the cell’s nuclei using fluorescent microscopy. As UV light is harmful for the cells, they have to be fixed first in order to avoid excess death during the assay. Our aim is to study changes that occurred due to treating cells with the acrylamide: cell division, cell apoptosis and amount of micronuclei.

Protocol

Fixation of the cells:

• Remove media
• Rinse 3 times with PBS (1 ml per well of 6-well plate)
• Add 1ml of 4% paraformaldehyde and 0,5% sucrose in PBS (per well of 6-well plate

Note: Paraformaldehyde is toxic! Always use gloves, safety glasses and work under the hood while using solution.

Staining with Hoechst:

• Remove the fixative with aspirator
• Rinse 3 times with PBS
• Add 1 ml of 10.000 times diluted stock solution of Hoechst
• Incubate 30 minutes in darkness at 37°C
• Remove stain
• Rinse once with PBS then add 1 ml of PBS

Preparing a microscope preparations:

• Prepare microscopic slides
• Place a drop of mountant in the middle of the slide
• Carefully place the round glass slide on the droplet (remember to put the slide side with cells to mountant down, so the cells stay immersed)
• Remove excess liquid

Observations:

Once the microscope preparations are ready it is possible to examine them using fluorescent microscope. Hoechst dye emits blue fluorescence light at around 461 nm while excited by ultraviolet light at around 350 nm.

Micronucleus assay

As we would like to measure the genotoxity of acrylamide, we’ve decided to implement micronucleus assay. This test belongs to the most popular and widely used methods of searching for potentially mutagenic substances.

The assay bases on formation of the micronuclei, which are the erratic nuclei formed during the anaphase due to mutagen exposition. They may originate both from acentric chromosome fragments or whole chromosomes unable to migrate properly.

The modifications in chromosomes structure, observed during the test, indicate the genotoxic effect of tested substance.

In our lab we would treat different cell lines with various acrylamide concentrations and after dyeing with Hoechst we would count the micronuclei. To examine the mutagenic effect of acrylamide we would use suitable statistical tests.

AlamarBlue assay

To indicate cell viability, we have decided to implement Alamar Blue assay. It bases on the ability of living cells to convert nonfluorescent resazurin to the highly fluorescent molecule- resorufin. While entering the cells, resazurin is being reduced to resorufin and the bright, red fluorescence is to be observed. Viable cells are continuing to convert resazurin to resorufin, so the strength of fluorescence increases proportionally and may be used to measure viability of the cells.

Protocol:

1st day Preparation of the 96-well plate:

• Adding 80µl media with 10.000 cells to each well
• Adding 20µl of properly concentrated acrylamide to each well
  • Row 1 and 2 -> no acrylamide: control
  • Row 3 and 4 -> 1mM acrylamide (1,76µl of acrylamide + 350 µl of PBS)
  • Row 5 and 6 -> 2mM acrylamide (3,52µl of acrylamide + 348,48 µl of PBS)
  • Row 7 and 8 -> 3mM acrylamide (5,28µl of acrylamide + 346,72 µl of PBS)
  • Row 9 and 10 -> 4mM acrylamide (7,04µl of acrylamide + 344,96 µl of PBS)
  • Row 11 and 12 -> 5mM acrylamide (8,8µl of acrylamide + 343,2 µl of PBS)

2nd day

• Adding 10µl of AlamarBlue to each well in rows A and B (24 hours incubation)
• Incubate for 2 hours at 37°C, protected from light, in a cell culture incubator
• Reading absorbance at 570 nm

3rd day

• Adding 10µl of AlamarBlue to each well in rows C and D (48 hours incubation)
• Incubate for 2 hours at 37°C, protected from light, in a cell culture incubator
• Reading absorbance at 570 nm

4th day

• Adding 10µl of AlamarBlue to each well in rows E and F (72 hours incubation)
• Incubate for 2 hours at 37°C, protected from light, in a cell culture incubator
• Reading absorbance at 570 nm

5th day

• Adding 10µl of AlamarBlue to each well in rows G and H (96 hours incubation)
• Incubate for 2 hours at 37°C, protected from light, in a cell culture incubator
• Reading absorbance at 570 nm

Flow Cytometry

Preparation of the samples

• Vacuum off media with floating cells and place it in different 15ml falcons.

• Rinse each well of 6-well plate with 2 ml of PBS. Vacuum off PBS

• Add 0,5 ml of Trypsin and 1 ml of PBS to each well.

• Incubate the plate for 10 min at 37°C

• Add 1 ml of media to each well to neutralize Trypsin

• Suspend the cells, then pipette them off and add to previously prepared falcons.

• Take a 50µl sample to perform a cell count

• Centrifuge for 4 min at 400 RPM

• Aspirate supernatant and suspend cells in PBS (1mln cells/1 ml)

• Transfer 300 µl of suspension to the test tube

• Centrifuge for 4 min at 400 RPM

• Aspirate supernatant and suspend cells in 100µl of binding buffer

• Add 4 μl of Annexin V or 5 μl of PI

• Incubate for 20 min at room temperature, protected from light

• Add 400µl of binding buffer, suspend, and transfer to test tubes used in your cytometer

• Analyze by flow cytometry