Part:BBa_K4719016

phaC

Introduction

Vilnius-Lithuania iGEM 2023 team's goal was to create synthetic biology tools for in vivo alterations of Komagataeibacter xylinus bacterial cellulose polymer composition. Firstly, we chose to produce a cellulose-chitin copolymer that would later be deacetylated, creating bacterial cellulose-chitosan. This polymer is an easily modifiable platform when compared to bacterial cellulose. The enhanced chemical reactivity of the bacterial cellulose-chitosan polymer allows for specific functionalizations in the biomedicine field, such as scaffold design. As a second approach, we designed indigo-dyed cellulose that could be used as a green chemistry way to apply cellulose in the textile industry. Lastly, we have achieved a of bacterial cellulose and polyhydroxybutyrate (PHB) composite, which is synthesized by K. xylinus.

We produced bacterial cellulose - PHB composite by introducing PHB synthesis operon into K. xylinus BBa_K4719017. The bacteria simultaneously produce both polymers, which are combined into the same material during the purification process.

Usage and Biology

PhaC is a gene encoding poly-β-hydroxybutyrate polymerase. The function of this protein is to polymerize (R)-3-hydroxybutyryl-CoA to create polyhydroxybutyrate (PHB), which consists of thousands of hydroxybutyrate molecules linked end to end. This is the last step of PHB synthesis.(1). PHB is a biodegradable thermoplastic, that naturally occurs as storage compounds in bacteria when limited by the lack of nutrients other than carbon. This gene was cloned from polyhydroxybutyrate synthesis operon in the plasmid pBHR68 (2).

References

1.Peoples, O.P. and Sinskey, A.J. (1989) ‘Poly-β-hydroxybutyrate (PHB) biosynthesis in alcaligenes eutrophus H16’, Journal of Biological Chemistry, 264(26), pp. 15298–15303. doi:10.1016/s0021-9258(19)84825-1.
2.2.Spiekermann, P. et al. (1999) ‘A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds’, Archives of Microbiology, 171(2), pp. 73–80. doi:10.1007/s002030050681. poly-β-hydroxybutyrate polymerase

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 769
  Illegal PstI site found at 1342
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 769
  Illegal PstI site found at 1342
  Illegal NotI site found at 145
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 587
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 769
  Illegal PstI site found at 1342
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 769
  Illegal PstI site found at 1342
  Illegal NgoMIV site found at 198
  Illegal NgoMIV site found at 313
  Illegal NgoMIV site found at 547
  Illegal NgoMIV site found at 859
  Illegal NgoMIV site found at 1138
  Illegal NgoMIV site found at 1551
  Illegal NgoMIV site found at 1618
  Illegal AgeI site found at 286
  
• 1000
  COMPATIBLE WITH RFC[1000]