Part:BBa_K5338006

FastPETase L1, Transcriptional Unit

This transcriptional unit is made of a J23101 promoter (BBa_J23101), BBa_B0034 RBS, FastPETase (BBa_K5338000), 3x stop codon, and a rrNB (BBa_B0010) terminator. This transcriptional unit was implemented into a pCA odd-1 backbone. PETase is a protein isolated from Ideonella Sakaiensis, that is found to degrade PET plastics into PET and MHET monomers. This was computationally optimized to create FastPETase, which was designed to be more efficient and heat-tolerant. Our team developed computational models for FastPETase, and simulated its stability under different temperature conditions.

The FastPETase amino acid sequence was obtained from NCBI, where it was published by Nazirov,M.M., Qobilov,F.B. and Khalilov,I.M. We then independently optimized the corresponding codons for E.Coli.

In this plasmid construct, we successfully implemented our FastPETase codon sequence (BBa_K5338000) into a L1 plasmid. This was done via Golden-Gate assembly (Loop assembly) with the five above listed L0 parts. The Golden Gate method simultaneously joins multiple DNA fragments into a single plasmid, in our case, the PCA odd-1 backbone (Pollak et. al). The promoter (BBa_J23101) was inserted in the A-B, the RBS (BBa_B0034) was inserted in the B-C, and the terminator (BBa_B0010) was inserted in the D-F “slot.” This L1 plasmid was successfully transformed into E.Coli (confirmed via Colony PCR and Gel Electrophoresis), which was implemented into a culture of M9 minimal media with PET plastics. The FastPETase gene sequence has a signal tag that allows for extracellular secretion. Degradation of the PET plastic can be measured via FTIR Spectroscopy.

Future goals: Current attempts are to create a Level 2 plasmid in conjunction with MHETase, and conjugate this into Alteromonas macleodii. This would allow the full degradation pathway to be implemented in Alteromonas and a corresponding artificial marine environment (ASW media). If this is not possible, we aim to implement FastPETase into a pRF backbone (this is currently in pCA, which lacks an oriT) and conjugate the L1 w/out MHETase into Alteromonas. However, current attempts to create a FastPETaseL1 plasmid with pRF backbone have failed.

FTIR Results are currently inconclusive.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 87
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal PstI site found at 87
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 87
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 87
  
• 1000
  COMPATIBLE WITH RFC[1000]