Team:HokkaidoU Japan/Project/Biocapsule

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Introduction

The aim of our project was to make a smart system for industrial production of high-value added macromolecules by using BioDevice function in E. coli. We call it, “Bio capsule” method. At the beginning, we expected to be able to make “Bio-capsule” only by employing a function of “Aggregation module”, however, resultant E. coli cluster was not sufficiently hard enough to be recovered on filter through nylon mesh filtration. However we accidentally found that co-expression of both “Plastic Producing Module” and “Aggregation Module” results in forming hard and heavy E. coli clusters which can be called real “Bio capsule” we expected.

Method

We used two kinds of compatible plasmid vector to make E. coli expresses both Ag43 and all enzymes required for P3HB production. We chose pSB1C3 plasmid vector, a high copy number plasmid vector containing replication origin from R- factor, for the expression of “Plastic Producing Module” to produce enough amount of bio-plastic. We chose pSTV28 plasmid vector that contains compatible replication origin to pSB series: the most popular plasmid vector in iGEM, for expression “Aggregation Module”. It is widely known that if there were two similar plasmids those containing same replication origin in single cell, they compete with each other for replication and only one of which can be amplified in E. coli.
(fig1 目下製作中) As a pilot experiment, we made E. coli containing plasmid for “Aggregation module” and plasmid for “Plastic producing module”. “Aggregation module” is induced by L-arabinose, since Ag43 is expressed under control of PBAD promoter. “Plastic producing module” used in the experiment in table 1 and 2 is expressed under control of original promoter of gene cluster encoding all enzymes required for P3HB production in R. eutropha.

Results

The aggregation is induced by the addition of arabinose for 1%, and production of P3HB is initiated by the addition of glucose for 2% as energy source, so adding both chemicals to the culture media initiates to start expression of both modules. Hard and heavy E. coli clusters were observed only in a presence of these chemicals as shown in Figure 2.