Team:NYMU-Taipei/Project/Enter

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Latest revision as of 17:46, 27 October 2013

National Yang Ming University

1 Entering of Bee. coli

Entering of Bee. coli

Introduction

In this part, the main purpose is to send Bee. coli into bees and make sure it can survive in the midguts. We chose Escherichia coli 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, this method could successfully transport the engineered Bee. coli into the bees’ midgut.

Background

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.

Materials

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

We successfully encapsulated the engineered E.coli( E.coli with GFP gene), and controlled its size under 0.1mm. Then we tried to transfer the engineered E.coli into bees midgut by adding some beads into their food.

Alginate structure	The structure of alginate capsule

CCD-curing pellet

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 drug-like product which is the mixture of sugar and microencapsulation of engineered E.coli. Therefore, we mixed the solution of microencapsulation with sugar cube and dry it. Then, we create the drug pellet which can cure CCD.

Experiments

Comparison of DH5 alpha and MG1655

We transformed red fluorescent protein into MG1655 and DH5 alpha, and then spread these bacteria onto the LB agar plates. After cultured at 37 degree for 16 hours, we calculated the number of colonies and found the transformation efficiency of DH5 alpha is about 100-fold higher than MG1655.

DH5 alpha	MG1655

Oxidative stress tolerance of MG1655

To know the oxidative stress tolerance of MG1655, we tested the susceptibility to H₂O₂ in MG1655.

Survival test

MG1655 cells were cultivated at 37 ˚C until the optical density (OD600) of the medium became 0.4~0.6
The culture was equally dispensed (1 ml each) into sample tubes, and then cells were treated with various concentrations of H₂O₂ (final concentrations were as follows: 10μM, 50μM, 100μM, 500μM, 1 mM, 5mM, 10mM, 50mM and 100mM) at 37 ˚C for 60 min
Testing the OD of each samples
Doing serial dilution on samples by mixing fresh LB medium with samples
20μl of the diluted sample was spread out on a LB plate (amp-) and incubated at 37 ˚C (overnight)

Results and discussion

Susceptibility to H₂O₂ in MG1655 shown in OD	Susceptibility to H₂O₂ in MG1655 shown in colonies

From these charts, the oxidative stress tolerance of MG1655 is higher than 5mM, and epithelial cells is only 0.05mM. In the other words, the tolerance to ROS of MG1655 is over 100-fold higher than cells in midgut. Therefore, MG1655 will survive even in the stress environment in the midgut.

Treated with 100mM H₂O₂ diluted 10⁴times	Treated with 50mM H₂O₂ diluted 10⁴times	Treated with 10mM H₂O₂ diluted 10⁵times	Treated with 5mM H₂O₂ diluted 10⁵times	Treated with 1mM H₂O₂ diluted 10⁶times

Treated with 500μM H₂O₂ diluted 10⁶times	Treated with 100μM H₂O₂ diluted 10⁷times	Treated with 50μM H₂O₂ diluted 10⁷times	Treated with 10μM H₂O₂ diluted 10⁷times	Treated with 0μM H₂O₂ diluted 10⁷times

Comparison table of MG1655 and DH5 alpha

Comparison	MG1655	DH5 alpha
Strain	K12	K12
Genotype	F-, lambda-, rph-1	-fhuA2 lac(del)U169 phoA glnV44 Φ80' lacZ(del)M15 gyrA96 recA1 relA1 endA1
Characteristics	Similar to wild-type	Designed for transformation
Transformation efficiency	>1x10⁸cfu/μg	>1x10⁶cfu/μg
Oxidative stress tolerance	<0.1mM	>5mM

Entering the bees' midgut

Getting into bees’ midgut

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

We dropped some solution with microencapsulation of E.coli on the sugar cube. After three days, we pulled out bees’ midgut and see if there was engineered E.coli. We also pulled out bees’ midgut from those who did not intake engineered E.coli and compared the result.

Products

left up: bees' gut , right up: no fluorescent light, left down: bees' gut after intaking our product, right down: lots of fluorescent signal

After the engineered E.coli was made into our pellet product, we wondered that if the engineered E.coli still work. We dissolved our pellet product in sugar water and fed our bees. After 3 days, we pulled out bees’ midgut and see if there was any engineered E.coli. The result show that there were still engineered E.coli, that was our product really work.

Outcome

The sugar cube with engineered E.coli in it.

We selected MG1655 as the chassis of our circuit, then successfully finding a way which can avoid the threat of immune system and ensure that Bee. coli won't spread into the environment while transporting Bee. coli into bees. Besides, once our chassis entered the midguts of bees, it is able to survive and fight against Nosema. We mixed the solution of microencapsulation beads with sugar pellet and dried it. That's what we called CCD-curing pellet.

Retrieved from "http://2013.igem.org/Team:NYMU-Taipei/Project/Enter"

@@ Line 1: / Line 1: @@
 {{:Team:NYMU-Taipei/Header}}
+=Entering of ''Bee. coli''=
 ==Introduction==
-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 ''Bee. coli'' into the bees’ midgut.
+In this part, the main purpose is to send ''Bee. coli'' into bees and make sure it can survive in the midguts. We chose ''Escherichia coli'' 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, this method could successfully transport the engineered ''Bee. coli'' into the bees’ midgut.
 ==Background==
-===Why did we choose MG1655===
+<!--
+===Why did we choose to use MG1655===
 [[File:NYMU_Bacteria_exist_in_honeybees'_midguts_naturally.png|thumb|300px|right|'''Bacteria exist in honeybees' midguts naturally'''<br>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]]
-#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.
+#The main reason we want to choose one of the substrain, MG1655, of ''E. coli'' K-12 strain as our carrier is ''E. coli'' K-12 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.
-#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.
+#The second reason for choosing  ''E. coli'' K-12 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.
 #Therefore, MG1655 is supposed to survive in the midguts after we send it into bees by encapsulation.
-#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.
+#However, the transformation efficiency of MG1655 is not as good as that of DH5 alpha, which is another substrain of K-12. 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.
 <div style="clear:both;"></div>
+-->
-===Why do we encapsulate ''Bee. coli''?===
-[[Image:NYMU-Taipei_alginateacid.png|thumb|300px|right|Structure of alginate polymer. α-L-guluronate sugar residues is shown on the left and β-D-mannuronate on the right side.<br> Source: http://en.wikipedia.org/wiki/Alginate]]
-The host immune system imposes a great threat on our engineered ''Bee. coli''. Besides we have to transported our engineered ''Bee. 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 ''Bee. coli''.
-To solve this problem, we have to cover the ''Bee. 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 ''Bee. coli''.
+===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''.
-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.
+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''.
 <div style="clear:both;"></div>
-===How to get capsule in===
+===Materials===
-We have considered many methods to get capsule 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 ''Bee. coli'' with the food of bees( sugar water) for that
+There are many different biomaterials for cell encapsulation. Among those biomaterials, we chose alginate as our encapsulation material for its abundance, excellent biocompatibility and biodegradability properties.
-===How to keep bees alive out of the hives===
+We successfully encapsulated the engineered ''E.coli''( ''E.coli'' with GFP gene), and controlled its size under 0.1mm. Then we tried to transfer the engineered ''E.coli'' into bees midgut by adding some beads into their food.
-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 ''Bee. coli'' into the sugar water, we could easily help the bees to intake ''Bee. coli''.
+{|
+|-
+! Alginate structure
+! The structure of alginate capsule
+|-
+| [[File:NYMU_alginate1.png.png|thumb|right|300px]]
+| [[File:NYMU_alginate_structure1.png|thumb|right|300px]]
+|}
+===CCD-curing pellet===
+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 drug-like product which is the mixture of sugar and microencapsulation of engineered ''E.coli''. Therefore, we mixed the solution of microencapsulation with sugar cube and dry it. Then, we create the drug pellet which can cure CCD.
+<div style="clear:both;"></div>
-==Experiment==
+==Experiments==
 ===Comparison of DH5 alpha and MG1655===
-We transform red fluorescent protein into MG1655 and DH5 alpha, then spreading on the LB plates. After cultured at 37 degree for 16 hours, we calculated the number of colonies and found the transformation efficiency of DH5 alpha is about 100-fold higher than MG1655.
+We transformed red fluorescent protein into MG1655 and DH5 alpha, and then spread these bacteria onto the LB agar plates. After cultured at 37 degree for 16 hours, we calculated the number of colonies and found the transformation efficiency of DH5 alpha is about 100-fold higher than MG1655.
 {|
 |-
@@ Line 47: / Line 61: @@
 ===Oxidative stress tolerance of MG1655===
-To know the oxidative stress tolerance of MG1655, we test the susceptibility to H<sub>2</sub>O<sub>2</sub> in MG1655.<br>
+To know the oxidative stress tolerance of MG1655, we tested the susceptibility to H<sub>2</sub>O<sub>2</sub> in MG1655.<br>
-'''< Survival test >'''
+====Survival test====
 #MG1655 cells were cultivated at 37 ˚C until the optical density (OD600) of the medium became 0.4~0.6
-#The culture was equally dispensed (1 ml each) into sample tubes, and then cells were treated with various concentrations of H2O2 (final concentrations were as follows: 10μM, 50μM, 100μM, 500μM, 1 mM, 5mM, 10mM, 50mM and 100mM) at 37 ˚C for 60 min
+#The culture was equally dispensed (1 ml each) into sample tubes, and then cells were treated with various concentrations of H<sub>2</sub>O<sub>2</sub> (final concentrations were as follows: 10μM, 50μM, 100μM, 500μM, 1 mM, 5mM, 10mM, 50mM and 100mM) at 37 ˚C for 60 min
 #Testing the OD of each samples
 #Doing serial dilution on samples by mixing fresh LB medium with samples
 #20μl of the diluted sample was spread out on a LB plate (amp-) and incubated at 37 ˚C (overnight) <br>
-'''< Result and discussion >'''
+====Results and discussion====
 {|
 |-
@@ Line 65: / Line 79: @@
 |}
-From these charts, we know the oxidative stress tolerance of MG1655 is higher than 5mM, and epithelial cells is only 0.05mM. In the other words, the tolerance to ROS of MG1655 is over 100-fold higher than cells in midgut. Therefore, MG1655 will survive even in the stress environment in the midgut.
+From these charts, the oxidative stress tolerance of MG1655 is higher than 5mM, and epithelial cells is only 0.05mM. In the other words, the tolerance to ROS of MG1655 is over 100-fold higher than cells in midgut. Therefore, MG1655 will survive even in the stress environment in the midgut.
 {|
@@ Line 123: / Line 137: @@
-===Encapsulation test===
+===Entering the bees' midgut===
-[[Image:NYMU_TAIPEI_encapsulation.jpg|thumb|300px|right|those are encapsulaition beads and the small green spot in it is E.coli]]
+====Getting into bees’ midgut====
-We carried out an experiment to see if microencapsulation of ''Bee. coli'' can really help our engineered ''Bee. coli'' enter bees’ midguts. We assume that only when engineered ''E .coli'' protected by the alginate membrane, it can be transported to bees’ midguts.
+[[Image:NYMU_TAIPEI_ENTERING!!.jpg|thumb|600px|right|left up: bees' gut , right up: no fluorescent light, left down: bees' gut after intaking ''Bee. coli'', right down: lots of fluorescent signal]]
+We dropped some solution with microencapsulation of ''E.coli'' on the sugar cube. After three days, we pulled out bees’ midgut and see if there was engineered ''E.coli''.
+We also pulled out bees’ midgut from those who did not intake engineered ''E.coli'' and compared the result.
+<div style="clear:both;"></div>
-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.
+====Products====
+[[Image:NYMU_TAIPEI_productentering.jpg|thumb|600px|right|left up: bees' gut , right up: no fluorescent light, left down: bees' gut after intaking our product, right down: lots of fluorescent signal]]
+After the engineered ''E.coli'' was made into our pellet product, we wondered that if the engineered ''E.coli'' still work.
+We dissolved our pellet product in sugar water and fed our bees. After 3 days, we pulled out bees’ midgut and see if there was any engineered ''E.coli''. The result show that there were still engineered ''E.coli'', that was our product really work.
 <div style="clear:both;"></div>
-[[Image:NYMU_TAIPEI_ENTERING!!.jpg|thumb|600px|center|left up: bees' gut , right up: no fluorescent light, left down: bees' gut after fed E.coli, right down: lots of fluorescent signal]]
+==Outcome==
-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’ midguts and see if there is any engineered ''E. coli'' with GFP. As we expected, ''E. coli'' only exited in the midgut of those who had intake sugar mixed with microencapsulation beads of ''E.coli''.
+[[Image:NYMU_TAIPEI_product.jpg|right|The sugar cube with engineered ''E.coli'' in it. ]]
+We selected MG1655 as the chassis of our circuit, then successfully finding a way which can avoid the threat of immune system and ensure that ''Bee. coli'' won't spread into the environment while transporting ''Bee. coli'' into bees. Besides, once our chassis entered the midguts of bees, it is able to survive and fight against ''Nosema''.
+We mixed the solution of microencapsulation beads with sugar pellet and dried it. That's what we called CCD-curing pellet.
 <div style="clear:both;"></div>
-==Brief Result==
+{{:Team:NYMU-Taipei/Footer}}
-After we proved that microencapsulation beads 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 beads of engineered ''Bee. coli''. So we mixed the solution of microencapsulation with sugar powder and dry it. Then, we create the drug powder which can cure CCD.
-{{:TeamNYMU-Taipei/Footer}}