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]