Dundee 2016

Proof of Concept

Bactifeed

Chicken Feed Experiments

In order to examine the proof of concept for our project experiments examining longevity of a bacteria coated feed product as well as experiments investigating the ability of this product to actually kill bacteria were set up.

Before any experimentation could begin the chicken feed obtained from Dodson & Horrel had to be sterilised in order to ensure that the tests we would carry out would be reliable. This was achieved by weighing out 200g of chicken feed and autoclaving this in a 500ml screw top flask. This sterile chicken feed was then stored at room temperature until required.

Inoculation of Chicken feed with bacterial strains

To give us a wide range of data from our testing we decided to use a variety of strains of bacteria to inoculate our feed. We chose truncated colicin Ia with SSp2 warhead, truncated colicin Ia and empty MG1655 E.coli cells. This would allow us to determine that our device was responsible for the killing, if any was observed.

A large overnight culture was grown for each of the previously mentioned constructs / strains and the absorbance’s recorded. These values were adjusted down an absorbance of 0.05 through diluting with PBS to result in a 900ml solution. The 200g of chicken feed was emptied into a sterile tray and the inoculated PBS/bacterial solution was poured into the feed. This process was carried out for the empty MG1655 E.coli as well as the truncated colicin Ia with SSp2 warhead. This mixture of feed and bacteria was left to mix on a shaker for 3 hours then left over night to dry in a sterile fume hood. In an attempt to deduce what inoculation method will produce the best results the same constructs / strains were also used in a 50 and 100ml agar solution in order to coat while acting as a protective coat.

When the bacterial coatings had fully dried the inoculated feed was transferred back into a sterile 500ml screw top flask. To investigate the longevity of the product some of the feed was stored in the fridge for weekly testing and some was transferred to an anaerobic tube and stored under nitrogen also for weekly testing.

Weekly longevity testing

On a weekly basis a sample of the stored chicken feed was taken and vortexed in a constant volume of PBS. A 1ml sample of the liquid was transferred to an Eppendorf tube and from here a tenfold serial dilution was carried six times of the stock solution and a 3ul aliquot of these dilutions plated out on the same plate. After growing the colonies overnight, a calculation was performed with the number of colonies present on the most dilute sample in order to obtain the cfu/g for the bacterial colonies. Through this method, we would be able to deduce the better method of storage for our product.

Killing experiments

To test if our product is capable of killing E.coli cells, 0.1% arabinose agar plates were made and empty MG1655 E.coli cells were grown until an absorbance of 0.1 was recorded. 50ul of this culture was plated out and a coated feed pellet from each construct / strain pressed into the agar. This process was repeated with the slight alteration of mashing the pellet in LB before adding this to the plate.

When the bacterial coatings had fully dried the inoculated feed was transferred back into a sterile 500ml screw top flask. To investigate the longevity of the product some of the feed was stored in the fridge for weekly testing and some was transferred to an anaerobic tube and stored under nitrogen also for weekly testing.

Results

The following data was recorded over five weeks and used the methods described above to test the number of surviving cells on a weekly basis.

From here we could plot the data in order to obtain trends which would inform us about the ideal storage conditions for our construct.

LEGEND**Figure1. Shows the plotted data for the Pcol Ia-ssp2 coated feed, inoculated with 50 ml agar stored in the fridge and in nitrogen. **** MAIN TEXT *** From the plot we can see that the number of cells in the construct stored in the fridge has a massive peak at week 4, this may be due to the presence of mould starting to grow on the product. The same construct stored in nitrogen had a much more constant level of cells and no mould was present in these conditions.

LEGEND*** Figure 2. Shows the ploted data of empty MG1655 cells inoculated with 100 ml agar. MAIN TEXT *** From the data shown here we can see that the feed stored in refrigerated conditions continued to grow and although more cells did remain in the product, the product again went mouldy after 4 weeks. The same experiment stored under nitrogen gave a gradual decrease in cells, which would be expected and again, no mould was found to be present for the duration of testing.

LEGEND**** Figure 3. Shows the Pcol Ia-ssp2 coated feed inoculated with a 1 litre PBS solution. MAIN TEXT *** From the data presented here we can see that the number of cells in the fridge stored feeds stays roughly constant. The same test stored in the nitrogen gave a gradual decrease in cells which was to be expeted. This ensures that no unwanted growth was occurring.

MAIN TEXT ***From this data we can see that again the refrigerated feed managed to keep a constantly high number of cells however, some growth was still occurring. The nitrogen stored feed only showed growth in the final week of testing.

Conclusion

To test if our product is capable of killing E.coli cells, 0.1% arabinose agar plates were made and empty MG1655 E.coli cells were grown until an absorbance of 0.1 was recorded. 50ul of this culture was plated out and a coated feed pellet from each construct / strain pressed into the agar. This process was repeated with the slight alteration of mashing the pellet in LB before adding this to the plate.

Through the testing carried out here we can conclude that the optimal storage conditions for our product would be under nitrogen. This is due to the fact that too much unwanted growth was present in the refrigerated feeds which could result in making the livestock ill. This is also backed up by the longer shelf life of the nitrogen stored feed.

ACME Stomach Masher

The science working in the lab is great but we wanted to ask what would happen when our device is taken outside of the lab but because we cant bring our device out of the lab we brought the outside in.

We designed a piece of hardware, (ACME Stomach Masher) which would simulate a part of the intestinal tract, which our Colicin producing bacteria in the BactiFeed would be exposed to: the stomach. The stomach exerts mechanical stress, amongst other stresses, on the contents of the stomach. To prove that the BactiFeed we designed would survive the harsh mechanical stress we passed samples of BactiFeed through the ACME Stomach Masher. The Stomach masher uses a motorized pulley system to move a zip-lock bag of contents through the rollers taken from recycled printers. All parts of the hardware were taken from recycled or donated materials.

Figure 3 shows the video of the experiment carried out in our lab. The stomach masher may also be operated manually to save on energy and therefore create a more environmentally use of it. In the video we show a combination of motor force and manual utilization.

As can be seen from figure 4, the hardware sufficiently mashes up our samples, any liquids may be added to the sample such as adjusted pH media to enhance the stomach-simulated environment. The masher may also be applied to other uses, which require mechanical stress testing.

Figure 5: Western blot of experiment carried out by Stomach Masher. No GFP observed when bound by anti-GFP antibody.

The stomach masher experiment (for protocol please see the Protocols page) was repeated three times, once with total lysate, once with pelleted lysate and then once with clarified lysate. Western blots were then run to see whether we could detect any GFP with the anti-GFP antibody. No GFP was detected, this could be because the conditions were at a pH of 4 and the optimum for the promoter was between 5 and 6.

We also used this device to test parts sent to us by UCL 2016 IGEM team, so the hardware has aided in our collaborations!

To find out more about how the ACME Stomach Masher was made please view our Hardware page