Difference between revisions of "Team:Oxford/Test/Beads"

Aim

To improve on last years Oxford iGEM team biobead design, using agarose to make up beads and then coating them with a ethyl acetate and polystyrene mixture.

Method

In a fume cupboard, break up a standard (polystyrene) petri dish into small pieces and dissolve in the minimum amount of ethyl acetate.

Prepare a 150 mL 1.5 % Agarose solution in beaker with screw cap. Microwave on high for 2 minutes and then cool to 40 °C in water bath. When microwaving ensure the screw cap is placed on loosely.

Remove agarose solution from water bath and bring to laminar airflow cupboard. Pour the agarose into petri dish to a depth of 1cm. Allow to set; this should take roughly 15 minutes.

Still under laminar flow, use an autoclaved 1 cm diameter hole borer to core out the required number of identical agarose cylinders and place in a second petri dish and leave to dry.

Bring uncoated ‘beads’ to fume cupboard. Using autoclaved needles, pick up individual beads and dip in ethyl acetate-polystyrene solution, stand each bead on a needle upright in fume cupboard on blob of blu-tac to allow ethyl acetate evaporation and the coating to set. When the coating is almost set the beads should be able to be handled through gloves without damaging the coat. Remove needle and mould the polymer coat over the needle hole, sealing them.

Control of substances Hazardous to Health (COSHH) Assessment

Agarose

• Not a hazardous substance

Ethyl Acetate

• H225 – Highly flammable liquid and vapour.
• H319 – Causes serious eye irritation.
• H336 – May cause drowsiness or dizziness.
• P210 – Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking.
• P261 – Avoid breathing vapours.
• P305+P351+P338 – IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do so. Continue rinsing.

Ethyl acetate poses the biggest safety hazard in this experiment so therefore do all steps involving it in the fumehood. Be careful of cutting self when breaking up the Petri dish, as the pieces can be sharp.

Write Up

150mL of 1.5% agarose was made up using 2.25g of agarose powder and 150 mL of MilliQ.

In a fumehood, one standard petri dish was broken into small pieces and placed into a 500mL beaker. To this, 50 mL of ethyl acetate was added along with a stirrer bar and the mixture was left to stir for 10 minutes. After 10 minutes the petri dish was still not dissolved so a further 10 mL of ethyl acetate was added and left to stir again.

The agarose was then poured into a petri dish to a depth of 1cm and left to set for 15 minutes. After this was set, using an eppendorf tube with the lid cut off cylinders of agarose were cut out and placed into a second petri dish.

After the agarose was left to dry it was taken to the fumehood, to be coated. The agarose beads were placed onto the end of pipette tips and then dipped into the ethyl acetate-polystyrene mixture. These were then left to dry standing up on the pipette tips.

The beads were left to dry overnight and it was found that the coating did not stick sufficiently to the agarose.

Findings

• Beads are far too big to fit into a catheter, but could be used in larger pipes.
• It is very hard to get an even coating of the ethyl acetate-polystyrene mixture around the bead and it would tend to slide off.
• Ethyl acetate-Polystyrene mixture was difficult to clean up.
• Is the coating actually porous?

Aim

Try and make some beads out of alginate instead of agarose and see if we can make them smaller and more suitable to be placed inside a catheter.

Method

Make the 30ml of 1.2% alginate solution in dH2O with 0.9% NaCl, add 0.36g of alginate slowly to hot water followed and add a magnetic stirrer bar. Then leave to stir until all solid is dissolved.

To compare, make up 30ml of 1.2% alginate solution in dH2O. Add 0.36g of alginate slowly to hot water and add a magnetic stirrer bar. Then leave to stir all solid is dissolved.

Transfer this mixture in a 2mL syringe with a needle attached and drop the mixture into a solution of 0.1M calcium chloride. Vary the size of the syringe and height from which you drop and record the effect on the beads. Leave the beads in the CaCl2 solution for 5 minutes and then filter the beads out.

Control of substances Hazardous to Health (COSHH) Assessment

Sodium Aliginate

• Not a hazardous substance

Calcium Chloride Solution (0.1M)

• H319 – Causes serious eye irritation
• P305 + P351 + P338 - IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.

The calcium chloride poses safety hazards therefore when doing steps involving it make sure you are wearing safety goggles and gloves.

Write Up

Sodium Alginate, mass = 0.36g

Milli-Q Water, volume = 30mL

Used hot plate to heat the water. Then while stirring slowly added the sodium alginate. The mixture was left to stir. Once the solid had all dissolved the mixture was a gloopy gel.

Using a 5mL syringe without a needle attached the sodium alginate solution was dropped into 0.1M calcium chloride solution, which was already made up in the lab.

The beads were then filtered using filter paper and placed into a petri dish and the process was then repeated using a syringe with a needle.

Without a needle

• Inconsistent size
• Fewer beads produced

With a needle

• Consistent size
• More beads produced
• Preferred method

Tests

Beads from the with needle trial were placed into 3 eppendorf tubes. One was placed at room temperature, one at 4 °C and one at -20°C.

Aim

Attempt to encapsulate cells inside of the sodium alginate beads. To test whether the cells are inside of the bead and to see if they are still alive we will use fluorescent bacteria and then measure the fluorescence of the beads under difference conditions.

Method

Take 1mL of cells in culture medium. Spin down in a centrifuge at maximum speed for 1 minute. Remove the culture medium. Re-suspend cells in 1mL of sodium alginate.

Make beads using the same method as above.

96-Well Plate

To attempt to see whether the cells are alive inside of the beads we will use a 96-well plate with different varieties of beads/medium/etc and then use a plate reader to measure the fluorescence.

	1	2	3	4
A	Bead with no bacteria	Bead with MG(-)	Bead with 20k MG	Bead with 20K DH5
B	Bead with no bacteria	Bead with MG(-)	Bead with 20k MG	Bead with 20K DH5
C	Bead with no bacteria	Bead with MG(-)	Bead with 20k MG	Bead with 20K DH5

Control of substances Hazardous to Health (COSHH) Assessment

Sodium Aliginate

• Not a hazardous substance

Calcium Chloride Solution (0.1M)

• H319 – Causes serious eye irritation
• P305 + P351 + P338 - IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.

The calcium chloride poses safety hazards therefore when doing steps involving it make sure you are wearing safety goggles and gloves.

In this experiment you are working with live bacteria therefore make sure everything used its sterile and you wear gloves.

Write Up

First 30mL of Sodium Alginate solution was made up using the protocol from 14/08/2015.

Then taking the 3 bacteria being encapsulated (MG(-), 20K MG, 20K DH5 a 1mL portion was placed into an eppendorf. These were then spun in a centrifuge at maximum speed for 2 minutes. The cells were then resuspended in 1mL of sodium alginate solution by pipetting up and down several times.

Beads were then made from the same method as in 14/08/2015.

To see whether any bacteria had been encapsulated the beads were viewed under a microscope. However due to problems focusing the microscope this was only completed for the first 2 beads.

Then the 96-well plate was made up and the fluorescences of each bead measured.

The wells that contained a bead that should have fluorescent bacteria encapsulated was much higher than the two controls. From this we could assume that the bacteria had been encapsulated inside of the bead however more analysis would be needed to study whether they were still alive.

Results

	1	2	3	4
A	164	152	1475	670
B	170	155	2082	794
C	163	155	1744	750

From the data we can make the assumption that the bacteria have been incorperated into the beads.

Column 1 contained no bacteria and has a low reading. Also column 2 contained bacteria that should not fluorsece which again had a low reading.

However column's 3 and 4 contained bacteria that should fluoresce, therefore we can conclude that the fluorescent bacteria are in the beads. This will be repeated over two more days to see if the results are consistent.