Preparation of cells with Different Optical Densities (OD)
We used this protocol to prepare the different OD600 cultures of bacteria that we were going to encapsulate in the hydrogels. We carried out this experiment to determine which OD of bacteria culture will be ideal for observing color change in the hydrogels. First, we decided to prepare bacteria cultures which contained our desired plasmid (aeBlue) of ODs of 1.0, 0.1 and 0.01. We repeated this same experiment with bacteria that contained pJUMP24-1A(sfGFP) to obtain cultures with ODs of 1.5, 1.0 and 0.75.
Note: The dilutions were achieved by using the equation: C1V1 = C2V2, where:
C1 = stock concentration, V1 = stock volume, C2= desired concentration, V2 = desired volume
Preparation of sodium alginate hydrogel encapsulated bacteria
This protocol was used to prepare the hydrogels. Sodium alginate hydrogels were prepared with Calcium Chloride as a cross linker. The Ca2+ from the solution interacts with the Sodium Alginate to create a three-dimensional gel structure. This allowed us to mold or create the desired hydrogel shapes.
The dilutions were achieved by using the equation: C1V1 = C2V2, where:
C1 = stock concentration, V1 = stock volume, C2= desired concentration, V2 = desired volume
3. For each of the tubes containing harvested cells from the cultures with different ODs from the Preparation of cells with Different Optical Densities, we resuspended the cells in 3mls of the different concentrations of sodium alginate. For example, the harvested cells from the bacteria culture of OD of 1.0 in three tubes, were resuspending in 3ml of concentrations of 10%, 5% and 2.5% sodium alginate respectively. This was repeated for the other tubes containing harvested cells from cultures with OD of 0.1 and 0.01. Below is a table with the description of the nine test categories.
Table 1 Test categories for hydrogel encapsulated bacteria containing aeBlue plasmid
4. After the resuspensions, a pipette or dropper was used to make spherical droplets of the various solutions in the 5% Calcium Chloride concentration. Nine different containers containing the 5% Calcium Chloride solutions were labelled according to the different concentrations of Sodium Alginate and the OD of the bacteria that was encapsulated by the Sodium alginate.
5. The hydrogels were left in the Calcium Chloride solution for 15 minutes to fully crosslink.
6. The hydrogel balls were then transferred to petri dishes lined with wet paper towels (wet paper towels with distilled water to keep the hydrogels moisturised) and placed in an incubator at a temperature of 37 degrees Celsius to monitor bacterial growth by observing at specific time intervals, colour changes in the hydrogel.
This test was done to check on the viability of bacteria in hydrogels after being allowed to grow in hydrogels over a period of 3 days. To determine the number of days that the bacteria can survive in the hydrogels, we used the following protocol.
1. One to three hydrogel spheres from the different test categories (cells at a particular OD resuspended in a particular concentration of Sodium Alginate) were placed in 5mls of LB media with antibiotic in different falcon tubes. There are nine different test categories as seen in Table 1.
2. Cultures were incubated with shaking at 37 °C and 140 rpm overnight.
3. The results were checked the next day. Cloudy cultures signify the growth of bacteria, indicating viable cells and vice versa. The O.D of the culture can be checked as well.
4. To clearly see the color, the overnight culture was centrifuged at 4°C for 10 minutes at 11,000 rpm. The supernatant was discarded leaving a pellet of cells left behind to also detect color changes.
After receiving our synthesized genetic parts, we used this protocol to assemble them.
1. We resuspended the Lithium Inducer, Arsenic Inducer and UV_T4 endolysin DNA part to a concentration of 10ng/µl each.
2. The reagents below were added to two tubes, one containing the Li-sensor and another the As-sensor DNA parts.
3. PCR was carried out at 37 °C 20 minutes, 37 °C 20 minutes 16°C 3 minutes for 15 times, 50°C 5 minutes, 80°C 5 minutes.
4. Transformation of the Li inducer plasmid and As inducer plasmid was carried out using the freshly prepared competent cells, BL21 and NEB 10 beta cells. Transformation was done following the general transformation protocol. Move to step 6.
5. Transformation was done again using the NEB5alpha commercial competent cells. The commercially competent cell protocol was used.
a. For each assembly, thaw a 50 µl tube of NEB 5-alpha competent cell on ice for 10 minutes.
b. Add 25 µl of the assembly reaction; gently mix by flicking the tube 4-5 times.
c. Incubate on ice for 30 min.
d. Heat shock at 42°C for 30 sec.
e. Place back on ice for 5 min.
f. Add 950 µl of room temperature SOC media. Incubate at 37°C for 60 min @250 r.p.m.
6. Plating was done with LB agar containing Kanamycin antibiotic.
7. Plated cells were incubated at 37 °C overnight.
8. Cell growths were inspected the next day under blue light and with the naked eye.
This is another golden gate assembly method that we used to corroborate our results.
1. The 25ul assembly reactions were set up as seen in the illustration above.
2. The reaction was mixed gently by pipetting up and down 4 times and transferred to the thermocycler and programed as follows: (5 min 37°C → 5 min 16°C) x 30 cycles followed by 5 min 60°C
3. Transformation was done using the NEB5alpha commercial competent cells. The commercially competent cell protocol was used.
a. For each assembly, thaw a 50 µl tube of NEB 5-alpha competent cell on ice for 10 minutes
b. Add 25 µl of the assembly reaction; gently mix by flicking the tube 4-5 times.
c. Incubate on ice for 30 min.
d. Heat shock at 42°C for 30 sec.
e. Place back on ice for 5 min.
4. 950 µl of room temperature SOC media was added and the mixture was incubated at 37°C for 60 min @250 rpm
5. LB agar plates containing kanamycin (for Pjump24-1A(sfGFP)) were warmed at 37°C for 15 min.
6. After the cells were mixed thoroughly by flicking the tube and inverting, 100 µl of the culture was spread onto each plate.
7. The plates were incubated overnight at 37°C.
pJUMP24-1A(sfGFP) digestion with Bsal-HFv2
This protocol was used to trouble shoot the results we got from the parts assembly. Seeing that the results we got were not as desired. We used several different BsaI-HFv2 enzymes for this digestion to ensure the plasmids were being cut to the right sizes. We also perform an insilico digestion of the pJUMP24-1A(sfGFP) using Benchling.
1. Set up reaction as follows per 50ul reaction.
i. Add 8.7ul of 114.5ng/ml pJUMP24-1A(sfGFP)
ii. Add 5ul of 10X rCutSmart buffer.
iii. Add 1ul of Bsal-HFv2
iv. Add 35.3ul of nuclease free water.
2. Carry out PCR at 37°C for 20 mins and 60°C for 20 sec.
3. After PCR, conduct Gel Electrophoresis.
This experiment was used to confirm if we had the right parts in the plasmids.
Preparation
1. Add 80 ml of 1X TAE buffer into a glass laboratory bottle.
2. Add 0.8g agarose powder to the TAE buffer to make 1% agarose.
i) Calculated using this equation.
Agarose (grams) = Gel desired (%) x Volume 1x TBE buffer (mL)
3. Microwave until the agarose is completely dissolved.
i) (Do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel.)
ii) * Be careful stirring, eruptive boiling can occur.
4. Let the agarose solution cool to 60 or70 degrees Celsius. Or until you can comfortably put your hand on the flask. * Make sure it does not solidify!
5. Add 5uL of SafeView™ Classic or SafeView™ DNA Gel stain to the cooled agarose solution.
6. Pour the agarose into a gel tray with the well comb in place.
i) Pour slowly to avoid bubbles which will disrupt the gel.
ii) Make sure that the comb is nearest to the black electrode (cathode), as the DNA migrates towards the red electrode (anode).
7. Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.
8. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
9. Fill gel box with 1xTAE (or TBE) until the gel is covered.
10. Carefully load your samples into the additional wells of the gel.
11. Run the gel at 120V for 30minutes.
12. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
13. Use a transilluminator or any device that has UV light to visualize the results.
1. Add loading dye to each of your DNA samples. DNA samples for each well should be about 10 - 100ng. This protocol uses NEB 6x purple loading dye. To calculate the amount of loading buffer needed, use 1ul of 6x loading dye: 5ul DNA sample. Loading buffer serves two purposes:
i. It provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated.
ii. It contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.
iii. a. Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can infer the size of the DNA in your sample lanes.
