Team:Calgary/Secretion

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Secretion

Overview

After the E. coli BL21 (DE3) engineered by the Synthesis subgroup has produced polyhydroxybutyrate (PHB), it is stored as intracellular granules that range in size from 60-80nm. This creates a challenging problem: efficient extraction of the desired PHB from inside the cell. During the early stages of our project, we explored many possible PHB extraction methods. Before deciding on using a PHB-secretion system, we evaluated the advantages and disadvantages of each system in terms of our space application.

Method:	Secretion	Chemical Extraction	Heat-Induced Lysis
Description:	Secretion of PHB via the Type I Hemolysin secretion pathway, which is endogenous to E.coli (Rahman et al., 2013).	Traditionally used solvent extraction, using chemicals such as chloroform and sodium hypochlorite (Hahn et al., 1994).	T4 lysis genes (holins and endolysins) under the control of Lambda promoter, pR with thermosensitive repressor proteins, cI587. When heated, lysis genes become activated.(George et al., 1987).
Advantages:	No chemicals are required PHB can be removed from media without killing cells Secretion is automatic Easier separation of PHB from media because there is minimal amounts of cellular debris Continuous production process	Efficient yields Simple	No chemicals required Lysis is automatic
Disadvantages:	Lower recorded yield than other methods Less characterized in literature than lysis mechanisms	Transport of chemicals to and from Mars is high cost Requires manual input of chemicals Lysis kills cells, (would need to ship stocks of our engineered E. coli to Mars) Cellular debris from lysis makes separation of PHB more difficult	Heating our fermenters on Mars would use huge amounts of power Lysis kills cells, (would need to ship stocks of our engineered E. coli to Mars Cellular debris from lysis makes separation of PHB more difficult

Type 1 Secretion

Our Secretion System

Our Construct

Experiments

Future Directions

Works Cited