Part:BBa_K4719018
pKARA_RT3 styrene monooxigenase for indigo synthesis in K. xylinus
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1226
Illegal SpeI site found at 37 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1226
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal SpeI site found at 37 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1226
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1226
Illegal SpeI site found at 37 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1226
Illegal SpeI site found at 37
Illegal NgoMIV site found at 92
Illegal NgoMIV site found at 134
Illegal NgoMIV site found at 503
Illegal AgeI site found at 689 - 1000COMPATIBLE WITH RFC[1000]
Introduction
Vilnius-Lithuania iGEM 2023 team's goal was to create a universal synthetic biology system for Komagataeibacter xylinus for in vivo bacterial cellulose polymer composition modification. Firstly, we chose to produce a cellulose-chitin polymer 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 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.
Colorful cellulose was made by introducing styrene monooxygenase pKARA_RT3 BBa_K4719018 to K. xylinus. This enzyme can metabolize indigo and its other derivatives into indigo dyes. Bacteria produce cellulose alongside pigments. Since they are not water soluble, the final product retains the color.
Usage and Biology
The function of this construct is to introduce indigo synthesis into K. xylinus. It was achieved by selecting styrene monooxygenase pKARA_RT3 capable of metabolizing indole and other substrates like 5-bromindoline, 7-nitroindole, 7-methylindole, 1,6,7,8-tetrahydrocyclopentan indole, from the growth medium to obtain colorful bacterial cellulose in one step.
Dyed bacterial cellulose has applications as an alternative to leather because of its material properties, low infrastructure needs and biodegradability. What is more, the conventional process of dyeing textiles is harmful to the environment. This problem can be solved with applying synthetic biology to produce already colorful material [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 to preserve Anderson promoter J23104 BBa_J23104, RBS BBa_B0034 and terminator BBa_B0015. pKARA_RT3 was assembled into the backbone by Gibson assembly.
Experimental characterization
Production of in situ dyed bacterial cellulose
In situ dyed bacterial cellulose is synthesized by K. xylinus grown in the Glucose Yeast Extract broth (GYB) while shaking at 180 rpm at 28°C, for 7 days. As a carbon source, we used 2% glucose. Substrates indole (0.5mM), 5-nitroindole, bromindoline (0.5mM), 7-nitroindole (0.25mM), 7-methylindole (0.25mM), 1,6,7,8-tetrahydrocyclopentan indole (0.25mM), 5 were added to the growth medium for dye production by pKARA_RT3.
K. xylinus, which was modified with a construct containing styrene monooxygenase pKARA_RT3, produced a range of indigoid compounds. As can be seen in Figure 1, some of the pigments were water-soluble, while others were contained in the structure of the bacterial cellulose. After purification, cellulose retained color (Figure 2).
References
1.Walker, K.T. et al. (2023) Self-dyeing textiles grown from cellulose-producing bacteria with engineered tyrosinase expression [Preprint]. doi:10.1101/2023.02.28.530172.| None |