## Introduction

Last year, Oxford's iGEM team was our university's first entry into the competition. They used beads to contain their bacteria and protect them from the surrounding environment, while allowing useful proteins to diffuse out of them. This year we improved on their design by using agarose to make up the beads and then coating them with an ethyl acetate and polystyrene mixture. This adds an extra layer of safety to prevent our bacteria from colonising the urinary tract.

We came up with the idea of improving on our previous team's project 8 weeks into the summer. Below is a notebook of the experiments we carried out.

### 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.

#### Without a needle

• Inconsistent size

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.

#### With a needle

• Consistent size
• 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.

### 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.

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 were 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

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

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

However columns 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.

### Aim

Repeat results from 17/08/2015 to check whether we get consistent results. To make a batch of beads that can be frozen and then tested for the bacteria viability at a later date.

### Method

Repeat protocol from 17/08/2015

Freeze a couple of each beads:

• No bacteria
• MG (-) (if we overnighted any, if not ask if we have an equivalent)
• 20K MG
• 20K F
• 20K DH5

### Control of Substances Hazardous to Health (COSHH) Assessment

This experiments has the same hazards as in 17/08/2015

### 96-Well Plate

In order to get a greater set of data than in 17/08/2015 the fluorescence of more beads to be tested.

### Write Up

30mL of sodium alginate made up using 0.36g of sodium alginate and 30mL of Milli-Q.

• No bacteria
• 20K MG
• 20K F
• 20K DH5
• MG(-)

The beads were then washed with Milli-Q and the fluorescence was measured.

The rest of the unused beads were then placed into the -20°C freezer to be tested at a later date.

### Results

Raw data was collected for the plate reader and then collaborated into a graph.

From the graph is can be shown that again the wells that contain beads encasing fluorescent bacteria have much higher fluorescences than the two control beads. Therefore this backs up the findings of yesterday that the bacteria is being encapsulated inside of the beads.

### Method

Make up one set of each type of bead listed below:

• No Bacteria
• MG(-)
• 20K MG
• 20K deltaF
• 20K DH5

Measure the fluorescence.

### Control of Substances Hazardous to Health (COSHH) Assessment

Same hazards as 17/08/2015.

### Write Up

1mL of each bacteria in LB was placed into an eppendorf and spun down at max speed for 2 minutes. The media was then swapped for M9 and diluted 1/20 in M9 in conical flasks.

• Cells = 1mL
• M9 = 19mL

However no MG(-) was overnighted so they could not be made into beads.

Therefore the layout of the 96-well altered to this:

These flasks were then left in the incubator at 37C.

OD measurements were taken at regular intervals and when the reading reached 0.4-0.7 the flasks were taken out of the incubator.

Beads made up from 2mL of the M9 and bacteria being spun down and resuspended in sodium alginate solution.

Fluorescence was measured in the same was as before.

### Results

Raw Data:

Results in agreement with previous two days.

### Method

Media swap and dilute to 1/20 in M9, leave in the incubator until the OD reads 0.4-0.7.

Make up beads using each of the bacteria.

Measure fluorescence and then freeze what is left.

### Control of Substances Hazardous to Health (COSHH) Assessment

Same hazard as 17/08/2015.

### Write Up

Media changed to M9 using 1mL of the overnight cultures from each bacteria and then diluted 1/20 in M9 in conical flasks. These flasks were then left in a 37C incubator.

The OD was measured at regular time intervals until they reached between 0.4-07.

The fluorescences was then measured according to before.

### Results

Raw data:

Third day of concordant results.

Place 1mL of LB + Chl into 5 test tubes. In the a test tube place one type of bead and then repeat with the other 4 test tubes.

Freeze any unused beads, labelled with the date.

### Control of Substances Hazardous to Health (COSHH) Assessment

Same hazards as 17/08/2015.

### Write Up

Media was swapped and diluted to 1/20 and then flasks left in the 37 incubator.

OD measured at regular intervals.

30mL of 1.2% sodium alginate was made up.

• Milli-Q, volume = 30mL
• Sodium alginate, mass = 0.36g

Beads of each type were made up trying to be as sterile as possible. The beads were not filtered out of the excess CaCl2 but instead it was poured off, this was to avoid any contamination from the filter paper.

The fluorescence was then measured.

5 eppendorfs of 1mL LB+Chl made up and a bead placed in each, left over the weekend.

### Results

Raw Data:

Experiment repeated for the 5 days in a row and consistent data gathetered.

### Update

Stop using 20K DH5, just takes too long to grow, it can be left for 7 hours and only reach OD measurement of 0.285. However it should reach 0.4 at least.

Beads left in the broth over the weekend did go cloudy in the end. Not the best results. Is the bead actually containing the bacteria or is this from contamination?

### Method

Media swap from LB to M9 using a 1/20 dilution.

Make up a set of beads of each type.

Measure the fluorescence.

### Control of Substances Hazard to Health (COSHH) Assessment

Same hazard as 17/08/2015.

### Write Up

Instead of giving up on DH5, a 1/10 dilution was made up to speed up time to reach log phase. Therefore check OD in 2 hours.

OD’s were measured at regular intervals until it reached between 0.4 - 0.7.

When the cells had reached log phase they were made into beads, the fluorescence was then measured as before.

The broth from 21/08/2015 was analysed under the microscope to see whether any bacteria was present. Unfortunately bacteria were present in the broth however it was not fluorescent so could either be contamination from the outside of the bead or bacteria from inside of the bead in stationary phase so not fluorescing. Therefore a method to sterilise the beads would be required. To do this the beads could be quickly dipped into ethanol and then stored in a sterile environemt.

### Results

Raw Data:

The experiment has now been reapted over 5 days and got consecutive results. Therefore we can assume that the bacteria has been encapsulated inside of the bead.

### Control of Substances Hazard to Health (COSHH) Assessment

Same hazard as 17/08/2015.

### Write Up

20K MG, 20K ∆F, and MG(-) were media swapped (1mL) into M9 and diluted 1/20, these flasks were then left in the 37C incubator. 20K DH5 was media swapped (1mL) into M9 then diluted 1/10 and the flask left in the 37C incubator.

The OD was measured at regular intervals and the flasks removed from the incubator when the OD had reached between 0.4-0.7.

The 4 different bacteria were encapsulated into Sodium Alginate solution and beads were made up.

The beads were then placed into a 96-well plate according to the layout:

Fluorescences measured using the GFP protocol.

### Results

Raw data that was collected from the plate reader. From this the graph was created in order to be able to analysis the data more effectivly.

From the results all of the beads that were frozen showed hardly any more fluorescence than the wells with no bacteria at all. Therefore we can conclude that freezing the beads would not be a suitable storage method.

The only beads that showed greater fluorescence were those that were made on the day.

There is a large peak for 19/08/2015 MG(-), this could be an anomaly or experimental error as all other beads with that bacteria have very little fluorescence.

### Control of Substances Hazardous to Health (COSHH) Assessment

Sodium Fluorescein:

• Not a hazardous substance

### Solutions to Make

From 0.02M stock solution of Sodium Fluorescein dilute to make solutions of 1µM and 5µM.

For 1µM solution:

Solution Volume (µM)
0.02M Sodium Fluorescein 25µL
Milli-Q 500mL

For 5µM solution:

Solution Volume (µM)
0.02M Sodium Fluorescein 125µL
Milli-Q 500mL

### Control of Substances Hazardous to Health (COSHH) Assessment

Sodium Fluorescein:

• Not a hazardous substance

Crystal Violet:

• H302 - Harmful if swallowed.
• H318 - Causes serious eye damage.
• H350 - May cause cancer.
• H410 - Very toxic to aquatic life with long lasting effects.
• P201- Obtain special instructions before use.
• P273 - Avoid release to the environment.
• P280 - Wear protective gloves/ eye protection/ face protection.
• P305 + P351 + P338 - IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
• P308 + P313 - IF exposed or concerned: Get medical advice/ attention.
• P501 -Dispose of contents/ container to an approved waste disposal plant.

### Calibration Curves

Sodium Fluorescein

Concentration (µM) Absorption
1 0.014
2 0.024
3 0.036
4 0.056
5 0.068
10 0.156
20 0.435
30 0.509
40 0.736
50 0.897

Crystal Violet

Concentration (M) Absorption
0.00000245 0.003
0.00001225 0.004
0.015 0.0000245
0.043 0.0001225
0.088 0.000245
0.456 0.001225
0.931 0.00245

### Experiment

• 2mL of 500µM Sodium Fluorescene
• 18mL Sodium Alginate Solution

Beads placed into 100mL (±5%) Milli-Q and 1mL portions removed every 10 minutes. The absorption of these fractions was then measured at 480nm. Experiment repeated 3 times.

### Data

Data collected and the averages of the absorption taken. The concentration of the solution can then be calculated using the calibration curve made earlier.

The data was the plotted on a graph of Concentration (µM) vs Time (min).

From the graph if is easy to see the rate of diffusion out the beads increases and then levels out when there is an equilibrium between the sodium fluorescence concentration in the beads and the concentration in the solution.

### Control of Substances Harzardous to Health (COSHH) Assessment

Same hazard as 02/09/2015.

Five beads were selected at random and used to calculate an average of the mass and diameter. The beads were assumed to be perfectly pherical and the diameter was measured 3 times for each bead and then an avarage of all 15 measurements taken.

From these measurements the density and volume of the beads could be calutated.

Volume = $\frac{4\pi&space;}{3}r^{3}$ where r is the radius of the bead.

Density = mass/volume

Measurement Average Value
Mass 0.0283g
Diameter 3.39mm
Volume 2.05x10-8m3
Density 138.53kg/m3

### Experiment

Experiment from 02/09/2015 was set up using Crystal Violet instead of Sodium Fluorescence and repeated 3 times.

• 2mL of 01% Crystal Violet
• 18mL Sodium Alginate

The results from these two days of experiments were then passed onto the modelers.

### Data

Data collected and the averages of the absorption taken. The concentration of the solution can then be calculated using the calibration curve made ealier.

The data was the plotted on a graph of Concentration (µM) vs Time (min).

From the graph if is easy to see the rate of diffusion out the beads increases and then levels out when there is an equilibrium between the sodium fluorescence concentration in the beads and the concentration in the solution.

### Experiment

Overnights (20K MG, 20K ΔF, 20K DH5α, MG(-)) had 1mL taken and media swapped to M9 then diluted 1/20. These flasks were then left in the 37˚C incubator until the OD reached roughly 0.1

1mL of cells in media was taken and the cells resuspended in 1.2% Sodium Alginate solution and made into beads.

The beads were then set up in a plate and placed into the plate reader for 20 hours at 37˚C with the fluorescence measured every 15 minutes.

As a test half of the fresh beads were dipped into ethanol in an attempt to sterilise the outside of the bead. However from the data we could see that the ethanol simply killed the cells.

## Conclusion

We successfully made the beads and showed that crystal violet dye diffused out of them. This data was essential for our modelling. We showed that we could encaptulate GFP-producing bacteria inside the beads and that the bacteria stayed alive in storage for at least twenty days.