In order to test the PHB synthesis rate in E. coli under anaerobic and aerobic conditions as well as the bacterial growth, E. coli BL-21 was transformed using the biobrick BBa_K934001 and was cultured in TB medium. Aerobic and anaerobic media were inoculated with pyruvate 50mM to simulate the real conditions under our bacteria would work. This quantity was used because is necessary to suppress the pyruvate dehydrogenase complex repressor (pdhR) ensuring the theoretical maximum expression rate of pyruvate dehydrogenase complex which allows the metabolism of pyruvate into acetyl-coA, the first substrate in PHB synthesis.

Each 24 hours we measured the bacterial growth of both media and after 72 hours the bacterial culture was centrifugate until get a pellet, which wasstored at -20°C during 72 hrs, after this time chloroform was added to get the PHB. The measure of PHB production showed a production of 25mg in media with oxygen and 90mg of PHB under anaerobic conditions.

**Figure 1:** PHB production under aerobic and anaerobic conditions

**Figure 2:** Bacterial growth under anaerobic and aerobic conditions

Conclusion

The result showed that PHB in E. coli works better under anaerobic contions even if the number of microorganism is less tha the aerobic conditions. We think this biobrick would work fine in our theoretical conditions where there are no oxygen and exist a high concentration of pytuvate, in our experiment we confirm this molecule enhances the PHB production because under high concentration it repress genes related to aerobic pathways, in this conditions the pyruvate obtained is metabolized in PHB.

Team:BUAP Mexico/Experiments

Here you can download some experiments that we did.

Aanerobic conditions test

Conclusion