Team:ZJU-China/Project/Safety/CaCO3Shell

Safety part I : Encapsulation of Living E.coli with CaCO3 / Ca3(PO4)2

Our atrazine killer is designed to move in the plate and in the soil, if applied to eliminate the atrazine in the environment, and thus we need to guarantee that it will not move around and pose potential threat to the environment. Here, we come up with an idea to encapsulate our engineered bacteria with CaCO3/ Ca3(PO4)2 on the purpose of constraining their movement before using and extending the preservation time.

As a biocompatible, stable and nontoxic shell, calcium carbonate and calcium phosphate have received increased attention in material sciences. The use of decomposable shell will not only prevent our engineered bacteria from posing potential threat to environment before they are designed to function ,but have little negative impact on the viability of the living organism.

CaCO3 / Ca3(PO4)2 spontaneously precipitates by mixing of two concentrated solutions of calcium and carbonate/phosphate. The precipitation leads to small, spherical and non-aggregated amorphous nano-precipitates, instantly formed upon mixing saturated salt solutions like CaCl2 and Na2CO3/ Na2HPO4. We use the similar conditions to produce CaCO3 and Ca3(PO4)2 shell published by Jennifer Flemke^[1] and Wang Ben^[2] respectively.

Protocol of Encapsulation of E.coli using CaCO3

1. 1 mL of overnight culture are added to a sterile reaction tube and centrifuged for 1 min at 12000 rpm.
2. The cells are washed 3 times with 0.33M CaCl2
3. Suspend the cells in 5 mL of 0.33M CaCl2 solution and incubate them under gentle stirring for 15 min.
4. A 5mL aliquot of 0.33M Na2CO3 is added under vigorous stirring for 30s.
5. Keep the whole suspension under stirring for 30 min for the precipitation of the CaCO3 microcapsules.

Protocol of Encapsulation of E.coli using Ca3(PO4)2

1. 1 mL of overnight culture are added to a sterile reaction tube and centrifuged for 1 min at 12000 rpm.
2. The cells are washed 3 times with 0.05M CaCl2
3. Suspend the cells in 5 mL of 0.05M CaCl2 solution and incubate them under gentle stirring for 15 min.
4. A 5 mL aliquot of 0.03M Na2HPO4 is added under vigorous stirring at the speed of 3 drops per minutes.
5. Keep the whole suspension under stirring for 30 min for the precipitation of the Ca3(PO4)2microcapsules.

To release the cells from the calcium capsules, the capsules are dispersed in 0.25M EDTA (pH=8) solution under constant shaking for 30min and afterwards washed twice with 0.25M EDTA(pH=8) and twice with 0.05M NaCl.

Reference

[1] Flemke J, Maywald M, Sieber V. Encapsulation of Living E. coli Cells in Hollow Polymer Microspheres of Highly Defined Size[J]. Biomacromolecules, 2012, 14(1): 207-214.

[2] [http://cdmd.cnki.com.cn/Article/CDMD-10335-1012310722.htm http://cdmd.cnki.com.cn/Article/CDMD-10335-1012310722.htm]