Team:NYMU-Taipei/Project/Enter

Get E.coli into the bees--- E.coli encapsulation ?

Abstract

In this part, the main purpose is to send Bee. coli into bees and make sure it can survive in the midguts. Therefore, we chose MG1655 as the chassis of our circuit, then incorporating MG1655 into alginate microcapsules, and fed bees with sugar water containing microencapsulation beads. According to our experiment, it could successfully transport the engineered E.coli into the bees’ midgut.

Why did we choose MG1655

Bacteria exist in honeybees' midguts naturally
Babendreier, D., Joller, D., Romeis, J., Bigler, F., & Widmer, F. (March 01, 2007). Bacterial community structures in honeybee 48 intestines and their response to two insecticidal proteins. Fems Microbiology Ecology, 59, 3, 600-610

• 1. The main reason we want to choose one of the substrain, MG1655, of E. coli K12 strain as our carrier is E. coli K12 strain exists in the midgut's of honeybees naturally. In addition, compared to nother substrain of k-12, MG1655 was selected for having few genetic manipulations from the archetypal E. coli K-12 strain, and it has already been well-studied as well.
  2. The second reason for choosing E. coli K12 strain is that what we utilize to repress the sprouting of Nosema (Mannosidase), sense the invasion of Nosema (ROS) and kill Nosema (Defensin and Abaecin) are harmful to the microbes. Notably, they are naturally produced in bees as well. Besides, the immune system of honeybee will prevent our E. kobee colonizing in the epithelial cells of guts. To solve these problems, we select MG1655 which naturally exists in bees.
  3. Therefore, MG1655 is supposed to survive in the midguts after we send it into bees by encapsulation.
  4. However, the transformation efficiency of MG1655 is not as good as that of DH5 alpha, which is another substrain of K12. On the other hand, the oxidative stress tolerance of MG1655 is much better than DH5 alpha theoretically because it's more similar to wild-type.

Ways to get E.coli in

We have considered many methods to get E.coli into Bees’ midgut, such as spraying the water mixed with microencapsulation of E.coli into the hives. Considering bees’ habits, we choose the way to mix microencapsulation of E.coli with the food of bees( sugar water) for that

device to keep bees alive out of the hives

We constructed a device to keep the bees alive when they were out of the hives. The device contained a cup and an eppendorf. The cup was the main part which bees active, with many pores on the top to exchange the air. The eppendorf is attached next to the cup, with a special straw to supply the sugar water. The sugar water in the eppendorf is the food of bees. By mixing the microencapsulation of E.coli into the sugar water, we could easily help the bees to intake the engineered E.coli.

way to detect the nosema

Nosema infected bees' midgut cells. So if bees are infected by nosema, we can detect spores in their midgut. We pulled out bees' midgut, and extract it. If we can find spores under the microscope, we conclude that the bees are infected by nosema. We use the similar way to check if engineered E.coli have entered bees' midgut.
To ensure this way is plosible, we constructed an experiment. We encapsulated the E.coli with GFP and RFP gene and mixed those beads into the sugar water, which is the food of our bees. After the bees died, we pull out the midguts of bees and search our engineered E.coli under the microscope. And we make the hypothesis that this way is feasible.

Encapsulation

Why encapsulation?

The host immune system imposes a great threat on our engineered E.coli. Besides we have to transported our engineered E.coli in the bees through bees’ digest system, which may contain many kinds of digestive solution with variable pH value that do harm to our engineered E.coli.

To solve this problem, we have to cover the E.coli with a membrane. And encapsulation is an easy way to reach the goal. Comparing to other approaches, cell encapsulation is easy to manipulate. So we choose cell encapsulation as the approach to cover the E.coli.

Material

Structure of alginate polymer. α-L-guluronate sugar residues is shown on the left and β-D-mannuronate on the right side.
Source: http://en.wikipedia.org/wiki/Alginate

There are many different biomaterial for cell encapsulation. Among those biomaterial, we choose alginate as our encapsulation material for its abundance, excellent biocompatibility and biodegradability properties.

experiment

We carry out an experiment to see if microencapsulation of E.coli can really help our engineered E.coli enter bees’ midgut. We assume that only when engineered E.coli protected by the alginate membrane, can it be transported to bees’ midgut.

Before the main experiment, we have to create microencapsulation of E.coli. Cell Encapsulation is technically achieved by dropping a mixture of cells and liquid alginate into a calcium chloride solution which solidifies the droplets, transforming them into the hydrogel beads. We should control beads’ size within the range of 20~100μm to ensure the beads can be intake by bees. We use a machine, which can help us achieve this goal.

those are encapsulaition beads and the small green spot in it is E.coli

Next we separated our bees into three categories, feed them with sugar mixed with microencapsulation of E.coli, sugar mixed with E.coli without encapsulation and sugar only. The E.coli is engineered with GFP gene, so we could find it easily under microscope. After three days, we pulled out the bees’ midgut and see if there is any engineered E.coli. As we expected, there is only E.coli in the midgut of those who had intake sugar mixed with microencapsulation of E.coli.

left up: bees' gut , right up: no fluorescent light, left down: bees' gut after fed E.coli, right down: lots of fluorescent signal

Products—Bee.coli

After we proved that microencapsulation of E.coli could be sent into midgut through bees’ digestion system, we came up with an idea that we can create a product which is the mixture of sugar and microencapsulation of engineered E.coli. So we mixed the solution of microencapsulation with sugar powder and dry it. Then, we create the drug powder which can cure CCD.

To prove that the engineered E.coli still work, we carried out following experiment. We make the sugar and microencapsulation of E.coli into dry powder, and test if the engineered E.coli can be sent into bees’ midgut after the powder dissolved in water. And through the experiment, we found out that this method really work.

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