Team:RHIT/Parts

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patekm@rose-hulman.edu
5500 Wabash Ave.
Terre Haute, IN 47803

To see more information about our parts, including some design considerations and the sources of our parts, please check out the pages on the registry.

Plasmid 1 Parts

• BBa_K2716000 - Optimized Double-Mutant PETase

  PETase is an aromatic polyesterase that breaks PET into MHET, as well as some terephthalic acid and BHET. The double-mutated optimized PETase contains a S238F/W159H mutation, allowing for a more efficient enzyme.
  
  

• BBa_K2716002 - MHETase

  MHETase breaks MHET into terephthalic acid and ethylene glycol.
  
  

• BBa_K2716100 - Complete Plasmid 1

  This composite part contains a promoter, a ribosomal binding site, the double-mutated optimized PETase, a secretion tag (pelB), MHETase, and a terminator. PETase is an aromatic polyesterase that breaks PET into MHET, as well as some terephthalic acid and BHET. The double-mutated optimized PETase contains a S238F/W159H mutation, allowing for a more efficient enzyme. PelB is a secretion tag that will allow the PETase enzyme to be secreted outside of the cell. This increases the efficiency of the PET breakdown. MHETase breaks MHET into terephthalic acid and ethylene glycol. The second secretion tag is added so that the MHETase enzyme will also be secreted.
  
  

• BBa_K2716101 - Promoter+RBS+pelB+PETase

  This composite part contains a promoter, a ribosomal binding site, the double-mutated optimized PETase, and a secretion tag (pelB). PETase is an aromatic polyesterase that breaks PET into MHET, as well as some terephthalic acid and BHET. The double-mutated optimized PETase contains a S238F/W159H mutation, allowing for a more efficient enzyme. PelB is a secretion tag that will allow the PETase enzyme to be secreted outside of the cell. This increases the efficiency of the PET breakdown. This composite is designed for the testing of PETase in a vector that does not previously contain a promoter nor a terminator. Therefore this would cause an overexpression of PETase, theoretically overstressing the cell to death. I.E. it's a killswitch.
  
  

Plasmid 2

• BBa_K2716004 - Promoter

  LacI-repressed pTrc promoter
  
  

• BBa_K2716102 - Complete Plasmid 2

  This composite part contains the sequence that outlines the metabolic cycle that turns ethylene glycol into malate, which is then used by the cell as a carbon source. This is through glycolaldehyde reductase turning ethylene glycol into glycoladehyde. Glycoladehyde dehydrogenase then turns this compound into glycolate. Glycolate oxidase turns glycolate into glyoxylate, which is then turned into malate by malate synthase.
  
  

• BBa_K2716200 - Glycolate Oxidase Subunit D

  The first of three subunits that make up glycolate oxidase. Glycolate oxidase transforms glycolate into glyoxylate.
  
  

• BBa_K2716201 - Glycolate Oxidase Subunit E

  The second of three subunits that make up glycolate oxidase. Glycolate oxidase transforms glycolate into glyoxylate.
  
  

• BBa_K2716202 - Glycolate Oxidase Subunit F

  The last of three subunits that make up glycolate oxidase. Glycolate oxidase transforms glycolate into glyoxylate.
  
  

• BBa_K2716203 - Glycolate Oxidase Composite

  This is a composite of three subunits which form glycolate oxidase when expressed. Glycolate oxidase transforms glycolate into glyoxylate.
  
  

  The Glycolate oxidase we submitted was cloned from /E. coli/ MG1655 and contains three subunits. It was inserted into the pETDuet vector for expression and was induced by 0.1and 0.2 mM IPTG at 30ºC for 20 hours. All three subunit of Glycolate Oxidase(GO) are shown in the SDS-PAGE. GO contains three subunits, D,E, and F, which weigh 55.5 kDa, 38.3 kDa, and 45.0 kDa respectively. Lane 1 was BL21(DE3) with pETDuet-GO, but without IPTG induction process. Lanes 2 and 3 were BL21(DE3) with empty pETDute vectors and contain 0.1 and 0.2 mM IPTG. Lanes 4 to 7 are BL21(De3) with pETDuet-GO. Lanes 4 and 6 were induced with 0.1mM IPTG, and lanes 5 and 7 were induced with 0.2mM IPTG. All the inductions were done at 30ºC.

  

• BBa_K2716003 - Terminator used in both plasmids

  Terminator for E. coli pSB1C3 backbone used in BBa_K2716100 and BBa_K2716102 composite parts.
  
  

<groupparts>iGEM18 RHIT</groupparts>