Difference between revisions of "Part:BBa K4719028"

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phaC-phaA-phaB-pKARA_RT3 operon for in situ dyed bacterial cellulose - polyhydroxybutyrate composite

Sequence and Features

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 composite of bacterial cellulose and polyhydroxybutyrate (PHB), 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 combined into the same material during the purification process. As an environmentally friendly way of plastic production, we thought of combining PHB synthesis genes with styrene monooxygenase pKARA_RT3 into one operon, allowing the synthesis of a self-dyeing plastic-like polymer.

Usage and Biology

This construct is a combination of a polyhydroxybutyrate synthesis operon (phaC, phaA, phaB) producing PHB along with bacterial cellulose, together with styrene monooxygenase pKARA_RT3 in K. xylinus. PHB is stored in bacteria intercellularly, while cellulose is secreted outside of the cell. Simultaneously K. xylinus produces indigoid pigments from added indole compounds as a substrate for styrene monooxygenase. To combine all materials into one composite washing procedure at boiling temperatures is required.

Ready dyed bacterial cellulose-PHB composite is an alternative to petroleum-based plastics. The advantage of this material is enhanced strength, resistance and accelerated rate of biodegradation [1].

Since polymer production occurs in K. xylinus requires a specific plasmid (pSEVA331-Bb) backbone for successful replication. We choose to use BBa_K1321313 as it was characterized by iGEM14_Imperial team as the most suitable synthetic biology tool for Komagateibacter species. We performed PCR of the plasmid eliminating mRFP in order to preserve Anderson promoter J23104 BBa_J23104, ribose binding site BBa_B0034 and terminator BBa_B0015. The construct was cloned by utilizing BBa_K4719018 as a plasmid backbone containing styrene monooxygenase pKARA_RT3, where PHB synthesis operon was assembled into the backbone by Gibson assembly.

Experimental characterization

Verification and transformation of the in situ dyed bacterial cellulose-PHB composite

Sanger sequencing revealed that pSEVA331-Bb-phaC-phaA-phaB-pKARA_RT3 did not contain any deleterious mutations and was successfully transformed into electrocompetent K. xylinus cells as seen in Figure 1.

Growth burden

In order to work with E. coli for designing constructs and testing synthetic biology systems, the growth burden of said synthetic biology tools has to be measured. We performed growth burden evaluation by measuring OD600 for five hours of modified and unmodified E. coli DH5α. The composite of in situ dyed PHB did not inhibit the growth of E. coli as seen in Figure 2.

References

1.Ding, R. et al. (2021) ‘The facile and controllable synthesis of a bacterial cellulose/polyhydroxybutyrate composite by co-culturing Gluconacetobacter xylinus and Ralstonia eutropha’, Carbohydrate Polymers, 252, p. 117137. doi:10.1016/j.carbpol.2020.117137.