Part II: Acetate gene knockout

Objective:

In our co-culture system, both microalgae and E. coli are utilized to produce PHB.
However, E. coli tends to convert a portion of carbon source into acetate as a by-product.
The accumulation of the acetate not only wastes the carbon sources but also inhibit cell growth and metabolism.
To increase the yield of the PHB of our Co-culture system, we decide to knockout the genes ackA-pta gene which is closely relate to the production of acetate in E. coli.

Part III: Construction of PhaBAC plasmid

Objective:

Wild-type E. coli does not naturally produce PHB.
This circumstance is due to E. coli lack the essential enzyme and pathway for its synthesis.
If we introduce PhaBAC system into the E. coli, then our E. coli also gain the ability to synthesize PHB as microalgae do.
By co-culturing two organisms that can produce PHB on their own, we expect the overall PHB production can increase and the consumption of carbon sources of the overall system will decrease.
Similarly, If we introduce this system into the microalgae, its production of PHB will also increase.
However, since microalgae is harder for transforming plasmid, we start with E. coli.