Part:BBa_K1692034

Pank + Imperial + Lysis

Using cell lysis to facilitate P(3HB) extraction from E. coli

Since bacteria that naturally synthesize P(3HB) use the plastic as an energy storage mechanism, the plastic is produced inside the cell and stays there. Currently, the isolation of P(3HB) from the cells and culture media is one of the main bottlenecks in the P(3HB) production process – and the main source of its high cost of approximately $4-6 USD [1]. Traditional isolation methods include the use of large amounts of chemicals that can be hazardous to human health and the environment, such as chloroform and sodium hypochlorite, or other problems such as large volumes of waste water produced or impurities in the polymers [1]. Since one of our team’s goals has been to reduce up-mass of materials on space flights, we wanted to minimize the amounts of harmful (and heavy) chemicals that would need to be brought up as payload to extract the P(3HB).

We sought to accomplish this by introducing a lysis device into the PSB1C3 plasmid that contained the phaCAB operon and the gene for pantothenate kinase. The lysis device was developed by the 2008 Berkeley iGEM team. The device contains T7 phage antiholin under a weak promoter, and T7 phage holin and endolysin inducible under (L)-arabinose. This lysis device was not reported to kill entire populations of E. coli, and we saw this as an advantage for a continuous plastic-production system. We would be able to cause part of the population to lyse and release its P(3HB) into the media, and the rest of the cells can continue to grow. The rest of the population would continue growing and producing plastic. The plastic can be recovered from the media by causing the granules to aggregate, and then requiring lower amounts of solvents and less time to complete the purification process [2]. We were able to sequence the gene and the sequencing result agreed with our gene schematic.

Note: We digested the Pank+Imp construct (BBa_K1692021) with PstI and SpeI and digested the 2008 Cal lysis system with PstI and XbaI and ligated the two constructs together.The device is not RFC[10] compatible due to the presence of an EcoRI site in the Pbad promoter. We did not notice this compatibility problem when we cloned the construct and thus our future goal is to use site-directed mutagenesis to make the gene biobrick compatible.

Functionality Test

Our first preliminary experiment was growing cell cultures containing the Pank-Imperial-Lysis (Pil) gene and the negative control strain onto L-arabinose (+) and L-arabinose (-) plate. Our streaking result showed that there were few Pil cells growing after induction by arabinose than not being induced.

Preliminary lysis experiment After inducing cell lysis with L-arabinose, we wanted to check whether there were viable cells remaining. These experiments were done at the end of the summer; if we had more time, we could have used another method of testing cell lysis, such as a beta galactosidase assay. To check quickly, we streaked the same volume of cells onto two halves of a plate to see which grew more. The two left halves show growth of 5uL of culture after lysis. The two right halves show our negative controls. The top one is cells from a culture that contained our plastic genes, but no lysis system; the bottom one is cells from a culture that contained the lysis system and the plastic genes but were not induced. We see more growth in the right two halves, which is promising although not conclusive.

A second preliminary experiment we did is having an overnight culture of Pil and the negative control, Pank-Imperial (Pi). We then induced the cultures with 1 M of L-arabinose the next day and measured their OD600 over a time period of four hours. Unfortunately, 3/4 of the Pil cultures did not grow and so we used the last Pil culture and induced it with arabinose. Therefore, there was no biological replicates of the Pil cultures while there were biological replicates of the Pi culture. Our result was promising and agreed with our initial preliminary data, in which we saw a sharp decrease in the OD600 in the Pil culture after induction with L-arabinose.

Induction lysis experiment We grew four overnight cultures for the Pil strain (P3HB+lysis) and four strains of the negative control, Pi (P3HB), strain. Unfortunately, only one of the four Pil strain grew so we induced that culture with L-arabinose. As for the Pi strain, we had two biological replicates induced with L-arabinose and not induced with L-arabinose. We noted that the Pil strain induced with L-arabinose exhibited a significant decrease in OD600 while the negative control continued to grow with or without L-arabinose. This indicated that L-arabinose induced cell lysis in the Pil strain while L-arabinose itself had no detrimental effect on cell growth.

References

[1] Jacquel, N. et al. Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochemical Engineering Journal 39 (1), 2008. 15 – 27.

[2] Rahman, A. et al. Secretion of polyhydroxybuterate in Escherichia coli using a synthetic biological engineering approach. Journal of Biological Engineering 7 (24), 2013.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]