Part:BBa_K808010

tphB: reaction from DCD to protocatechuate

Homology modelling of TphB. For simulation parameters http://2012.igem.org/Team:TU_Darmstadt/Modeling_Homologie_Modeling#TphB click here.

TphB is coding for the decarboxylating cis-dihydrodiol dehydrogenase from Comamonas testosteroni Kf-1. It catalyzes the second reaction in the terephthalic acid degration - the decarboxylation of DCD to protocatechuate. The [http://2012.igem.org/Team:TU_Darmstadt/Modeling_Homologie_Modeling#TphB homology modeling] suggests a homodimerization as described by the recent paper by Bains et al.
For further information, please visit the [http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism TU Darmstadt iGEM team wiki].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 155
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 202
  Illegal NgoMIV site found at 877
  Illegal AgeI site found at 939
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 76
  

Part Characterisation

Figure 1. Note the peak in GPC at about 32 minutes (about 64 kDa)

GPC confirmed the suggested homodimerization (Figure 1).

Uppsala 2022 improvement

The original part BBa K808010 was obtained via colony PCR from its original host Comamonas testosteroni Kf-1. This is a fast and easy approach to obtain a gene without having it synthesized. A drawback when using this approach is the variation of codon usage in different host organisms. This can cause problems in the protein expression when transferring the gene into a different host. To allow efficient work with TphB in Escherichia coli (E. coli) our team optimized the codons and added a c-terminal histidine tag to allow purification through nickel based affinity chromatography. Addtionally mutated all illegal restriction sites to make this part compatible with the BioBrick standard. With those improvements our team managed a very strong overexpression of tphB.

Figure 2. Overexpression at two different temperature (24 °C and 37 °C) and IPTG concentrations (0,1 mM and 1 mM) of TphB using pET24a expression vectors. IF3 was used as control for overexpression. The TphB band is very strong and spans over huge area the expected size is 33 kDa which corresponses to the lower end of the band. The IF3 band is expected to be around 23 kDa.

These improvements resulted in the basic part BBa_K4378999 and the composite part BBa_K4378000 which can be directly used for overexpression in a pET-Vector without the use of any other functional parts of the T7 expression cassette.

References

• Sasoh, M., E. Masai, et al. (2006). "Characterization of the terephthalate degradation genes of Comamonas sp. strain E6." Appl Environ Microbiol 72(3): 1825-1832.
• Fukuhara, Y., K. Inakazu, et al. (2010). "Characterization of the isophthalate degradation genes of Comamonas sp. strain E6." Appl Environ Microbiol 76(2): 519-527.
• Kamimura, N., T. Aoyama, et al. (2010). "Characterization of the protocatechuate 4,5-cleavage pathway operon in Comamonas sp. strain E6 and discovery of a novel pathway gene." Appl Environ Microbiol 76(24): 8093-8101.
• Schläfli HR, Weiss MA, Leisinger T, Cook AM. (1994)"Terephthalate 1,2-dioxygenase system from Comamonas testosteroni T-2: purification and some properties of the oxygenase component." J Bacteriol. 1994 Nov;176(21):6644-52.
• Bains, J., J.E. Wulff, and M.J. Boulanger, Investigating Terephthalate Biodegradation: Structural Characterization of a Putative Decarboxylating cis-Dihydrodiol Dehydrogenase. J Mol Biol, 2012.