1.-Recognition of the bacteria by Gram-dye
Upon analysing the liquid and plaques, we saw that the bacteria we had stored from a year ago had no contaminants. Therefore, it gave us a pink colour, which is called negative gram, indicating that our bacteria was, in fact, E. Coli.
Figure 1. Gram dye of E. Coli cells.
We achieved to create proper LB agar plates in order to grow our bacteria strains. We had intended to grow them by practicing on super nutrient media with a special pigment to recognize negative gram bacteria which would glow green after a proper colony formation.
Figure 2. Team member growing bacteria in agar plates.
To create our competent cells we add CaCl2 and MgCl2 to our cells. We got competent cells after many trials because our bacteria were not strong enough to survive the heat shock.
Figure 3. Team members making competent bacteria.
To transform the bacteria we subjected them to heat shock for them to be able to accept the plasmid more easily. The bacteria’s membrane will become more permeable and through osmosis the plasmid will enter the bacteria’s cells. We called this culture Hope!
Figure 4. Agar plate with our transformed culture, Hope.
Quantification of DNA after bacteria growth.
For quantification of the concentration of the DNA contained in our bacteria after transformation we elaborated a mini-prep protocol. After the continuous enzymatic steps, the final quantification of plasmid DNA was 87 ng/µl, measured using a nanodrop with infrared light.
In the gel electrophoresis we could observe stripes that showed the mass of our DNA. We were able to recognize and prove the length of our parts: both the BioBrick and the rest of the plasmid. The BioBrick had a real length of 1983 bp, and was at the level of the 2000 bp of the ladders. The rest of the plasmid had a total length of 1739 bp and indeed the complete plasmid has a real length of 3722 bp. The gel proved that the total plasmid length was higher than the highest stripe of the 1kb ladder.
Figure 5: Gel electrophoresis with both 1kb and 100bp ladders in the first and second columns and the trials for quantification of the length of each fragment (the BioBrick and the rest of the plasmid.
Figure 6. The ladder pattern that we used as reference to determine the mass of the DNA (Thermo Fisher Scientific Inc., 2015).
We did several trials of alginate pearls. The first day, they didn’t work out due to the concentration of CaCl2 we were using because it was way too permeable. However, in later attempts we tried with three different concentrations: 1 M CaCl2 and 2% of Alginate, 2.5 M CaCl2 and 2% alginate, 2.5 M CaCl2 and 4% alginate in order to find the putative concentration of CaCl2 and we finally reached the conclusion that, as can be seen in the image, the ones that worked best to retain the dye were the last ones, CaCl2 2.5 and 4% alginate.
Figure 7. Alginate pearls with different concentration of methyl violet. On the right, the first trial Alginate pearls with vegetal colorants.
Protocol 1 - Liquid culture media
Preparation of culture media (1L).
5 g of yeast
10 g of sodium chloride.
10 g of tryptone.
1.- Mix the aforementioned compounds and add water until the 1 L volume.
Protocol 2 - Electrophoresis
1.- Prepare 1L of TAE 10X (48.4g of Tris base[ tris (hidroxymethyl) amminomethane], 11.4 mL of glacial acetic acid (17.4 M) and 3.7 g of EDTA then fill with deionized water to 1 L.
2.- Prepare a 50 mL solution of 3% agarose using TAE buffer (without using water).
3.- Heat the gel in the microwave in intervals of 30 s, 20 s and 10 s until the agarose is completely dissolved, taking care that it does not boil so much.
4.- Let cool down the gel (55-60°C) and pour in a base with the comb already on its place to form the charging wells.
5.- Wait until the gel solidifies.
6.- Remove the comb and place the base with the gel inside the electrophoretic chamber.
7.- Add TAE buffer until the gel is covered.
8.- Take 5 uL of each sample from the PCR and 1 uL of charging gel in a new PCR tube, mix well.
9.- Charge the samples inside the wells of the gel, adding a weight marker.
10.- Close the electrophoretic chamber and run at 120 V during 30 min. Follow the displacement of the samples.
11.- Take out the gel from the chamber very carefully and place in the UV light to see the DNA strands.
Protocol 3 - Competent cell transformation
1.- (Split #1) Split the 1 L culture into four parts by pouring about 250 mL into ice cold centrifuge bottles. Harvest the cells by centrifugation at 3000g (~4000 rpm in the Beckman JA-10 rotor) for 15 minutes at 4ºC.
2.- Decant the supernatant and gently resuspend each pellet in about 100 mL of ice cold MgCl2. Combine all suspensions into one centrifuge bottle. Make sure to prepare a blank bottle as a balance.
3.- (Spin #2) Harvest the cells by centrifugation at 2000g (~3000 rpm in the Beckman JA-10 rotor) for 15 minutes at 4ºC.
4.-Decant the supernatant and resuspend the pellet in about 200 mL of ice cold CaCl2. Keep this suspension on ice for at least 20 minutes. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
5.- ( Spin #3) Harvst the cells by centrifugation at 2000g (~3000 rpm in the Beckman JA-10 rotor) for 15 minutes at 4ºC. At this step, rinse a 50 mL conical tube with ddH2O and chill on ice.
6.- Decant the supernatant and resuspend the pellet in ~50 mL of ice cold 85mM CaCl2, 15% glycerol. Transfer the suspension to the 50 mL conical tube.
7.- (Spin #4) Harvest the cells by centrifugation at 1000g (~2100 rpm in the Beckman GH-3.8 rotor ) for 15 minutes at 4ºC.
8.- Decant the supernatant and resuspend the pellet in 2 mL of ice cold 85mM CaCl2, 15% glycerol. The final OD600of the suspended cells should be ~200-250.
9.- Aliquot 50uL into sterile 1.5mL microfuge tubes and snap freeze by placing for a brief time in ice-cold isopropanol or with liquid nitrogen. Store frozen cells in the -80º C freezer.
Protocol 4 - Alginate Pearls
500 ml beaker
100 ml beaker
Eppendorf tube 1.5ml
Small magnetic stirrer
Burette 50 mL
Micropipettes 100 - 1000 l
Magnetic stirring grill
4% sodium alginate
2.5 M calcium chloride 20 mL
4.5 mg enzyme protein
1.- Prepare the solution of sodium alginate 4% m / v. Prepare 100 ml of the solution of calcium chloride (molar mass: 110.986 g / mol) 2.5 M. Mix …..ml solution of the protein / enzyme with the sodium alginate.
2.- Take a sterile burette 100ml, add the prepared and dropping a couple of drops from 10cm to a 100ml beaker of calcium chloride under constant stirring using a magnetic stirrer solution (do not touch the solution CaCl 2 with pipette!),
3.- Regular size pearl to taste (cutting the pipette bulb or increasing the distance of the fall) and make a 30-35 pearls
4.- Leave stirring constantly alginate beads in calcium chloride solution for 20 minutes approx. to ensure complete solidification
Protocol 5 - Preparation of Agarose Plates for Media Culture
Materials for medium:
1.5% (1.5g/100mL) Agar
20g/1000mL LB (See Protocol 1)
Add the LB in distilled water. Once dissolved, add the agar and stir continuously over a hot plate.
After the mixture is completely homogenous, cover the flask.
Autoclave the medium.
After the sterilization of the medium, while working under aseptic conditions, add aprox. 30mL of it into each empty, sterile Petri dish.
Wait for the medium to become gel, and use Parafilm or a similar material to shut tight the dishes to avoid contamination of the medium.
Protocol 6 - Media Culture on Plate
Agarose-ready plates (See Protocol 5)
50-100 uL of bacterial strain
Sterile glass spheres
Working under sterile conditions, micropipette 50-100 uL of bacteria into an agarose-ready Petri dish.
Spread the bacteria across the dish by using sterile glass spheres and tilting carefully the plate with them inside.
Remove the glass spheres and store the plate overnight at a temperature of 27º C.
Protocol 7 - One liter 50X stock of TAE
Tris-base: 242 g
Acetate (100% acetic acid): 57.1 ml
EDTA: 100 ml 0.5M sodium EDTA
Mix the aforementioned materials and add dH2O up to one litre.
Protocol 8 - Preparation of agarose gel for electrophoresis, electrophoresis.
1. Measure out 0.80 g of agarose powder
2. Mix the agarose with 100 ml of IX TAE in a microwavable flask (See Protocol 7).
3. Cover the flask and microwave for 1-2 minutes.
4. Cool down agarose solution for 5 minutes
5. Add a final concentration of 5 ug/mL Ethidium Bromide to the flask (! For safety reasons, we substituted the Ethidium Bromide with GelRed with the same concentrations).
6. Pour solution into casting tray
7. Place comb within the casting tray
8. Let casting tray sit for 5 minutes at 40C or 20-30 minutes at room temperature.
Protocol 9 - Plasmid purification
Notes before starting
Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000.
Add the provided RNase A solution to Buffer P1, mix and store at 2-8°C
Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume).
All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge.
1.- Pellet 1-5 ml bacterial overnight culture by centrifugation of >8000 rpm (6800 x g) for 3 min at room temperature (15-25°c).
2.- Resuspend pelleted bacterial cells in 250 lBuffer P1 and transfer to a microcentrifuge tube.
3.- Add 250 lBuffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4.- Add 350 lBuffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution will turn colorless.
5.- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6.- Apply 800 l supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30-60 s and discard the flow-through.
7.- Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30-60 s and discard the flow-through. Note: This step is only required when using endA+ strains or other bacterial strains with high nuclease activity or carbohydrate content.
8.- Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30-60 s and discard the flow-through. Transfer the QIAprep 2.0 spin column to the collection tube.
9.- Centrifuge for 1 min to remove residual wash buffer.
10.- Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 uL Buffer EB (10mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
11.- If the extracted DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
Protocol 10 - Enzyme Assay
Set up the reaction using the following scheme:
1) Determine the amount (total ug and total ul) of DNA to be digested.
2) Use the ug amount of DNA to determine how many enzyme units to use.
3) Determine how many ul of enzyme to use, using the enzyme concentration (we used 1uL of each enzyme, this will vary depending on the type of enzyme you are using. The enzymes we used were Xba I and Spe I).
4) Choose a total volume for the reaction. The enzyme volume must be 10% or lessof the total digest volume, so choose a reaction volume that is at least 10 times the amount of the enzyme volume added. Choose a total volume, the minimizes the addition of water.
5) Determine the amount of buffer to use. The buffers used in the reaction are usually 10x stocks, which means they must be 10% of the final volume.
6) Finish reaction set up by adding water as necessary.
7) Digest at optimum temperature for 60 minutes.
8) Run a gel to verify the reaction is complete! (See Protocol 8 and 2).
Protocol 11 - Gram staining
“Heat-fix” the slide with the specimen by passing it over a heat source, such as a flame, several times using a forceps. The slide should be passed very quickly through the flame and not be heated excessively. Place slide on the staining tray.
Flood the fixed smear with crystal violet solution (#1) and allow to remain for 1 minute.
Rinse off the crystal violet with distilled or tap water.
Flood the slide with iodine solution (#2). Allow to remain for one minute. • Rinse off the iodine solution with distilled or tap water.
Flood the slide with decolorizer (#3) for one to five seconds.
Rinse off the decolorizer with distilled or tap water.
Flood the slide with safranin (#4). Allow to remain for 30 seconds.
Rinse off the safranin with distilled or tap water.
Dry the slide on bibulous paper or absorbent paper and place in an upright position. Microscopically examine the slide for bacterial organisms under a 100X objective. Observe several fields on the slide for bacterial organisms. Describe the gram reaction of any organisms seen. Gram-positive bacteria stain deep violet to blue and gram-negative bacteria stain pink to red.