Part:BBa_K4719015

phaB

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

PhaB is a gene encoding acetoacetyl-CoA reductase. The function of this protein is to catalyse the NADPH-dependent reduction of acetoacetyl-CoA. This step is intermediate in polyhydroxyalkanoates(PHA) pathways (1). PHAs are biodegradable thermoplastics, that naturally occur 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.Kim J, Chang JH, Kim EJ, Kim KJ, (2014) ‘Crystal structure of (R)-3-hydroxybutyryl-CoA dehydrogenase PhaB from Ralstonia eutropha’, Biochem Biophys Res Commun, 443(3):783-8. doi: 10.1016/j.bbrc.2013.10.150. Epub 2013 Nov 6. PMID: 24211201.
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.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 478
  
• 1000
  COMPATIBLE WITH RFC[1000]