Part:BBa_K4719028
phaC-phaA-phaB-pKARA_RT3 operon for in situ dyed bacterial cellulose - polyhydroxybutyrate composite
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 4992
Illegal SpeI site found at 37
Illegal PstI site found at 824
Illegal PstI site found at 1397 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 4992
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal SpeI site found at 37
Illegal PstI site found at 824
Illegal PstI site found at 1397
Illegal NotI site found at 200 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 4992
Illegal BglII site found at 642
Illegal BamHI site found at 3039 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 4992
Illegal SpeI site found at 37
Illegal PstI site found at 824
Illegal PstI site found at 1397 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 4992
Illegal SpeI site found at 37
Illegal PstI site found at 824
Illegal PstI site found at 1397
Illegal NgoMIV site found at 253
Illegal NgoMIV site found at 368
Illegal NgoMIV site found at 602
Illegal NgoMIV site found at 914
Illegal NgoMIV site found at 1193
Illegal NgoMIV site found at 1606
Illegal NgoMIV site found at 1673
Illegal AgeI site found at 341
Illegal AgeI site found at 4455 - 1000COMPATIBLE WITH RFC[1000]
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. This composite allows 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 parts, the growth burden of said synthetic biology constructs 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.
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