Part:BBa_K4719018

pKARA_RT3 styrene monooxigenase for indigo synthesis in K. xylinus
Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1226
  Illegal SpeI site found at 37
  
• 12
  INCOMPATIBLE 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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1226
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1226
  Illegal SpeI site found at 37
  
• 25
  INCOMPATIBLE 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
  
• 1000
  COMPATIBLE 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 bacterial cellulose and polyhydroxybutyrate (PHB) composite, which is synthesized by K. xylinus.
This specific part was used for creation of colored cellulose. Color was introduced by styrene monooxygenase pKARA_RT3 BBa_K4719018 to K. xylinus. This enzyme can metabolize indigo and its other derivatives into indigo dyes. Bacteria produces cellulose alongside pigments. Since they are not water soluble, the final polymer retains the color. ==Usage and Biology== The function of this construct is to introduce indigo synthesis into K. xylinus. It was achieved by 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, a specific plasmid (pSEVA331-Bb) backbone for successful replication is required. We choose to use BBa_K1321313 as it was characterized by iGEM14_Imperial team as the most suitable plasmid backbone 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), were added to the growth medium for dye production by pKARA_RT3.

Figure 1: A - bacterial cellulose grown on 5-bromindoline (0.5mM). B - bacterial cellulose grown on indole (0.5mM). C - bacterial cellulose grown on 1,6,7,8-tetrahydrocyclopentan indole (0.25mM). D - bacterial cellulose grown on 5-nitroindole. E - bacterial cellulose grown on 7-nitroindole. F - unmodified bacterial cellulose.

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).

Figure 2: A - bacterial cellulose grown on 5-bromindoline (0.5mM). B - bacterial cellulose grown on indole (0.5mM). C - bacterial cellulose grown on 7-nitroindole D - bacterial cellulose grown on 7-methylindole (0.25mM). E - bacterial cellulose grown on 1,6,7,8-tetrahydrocyclopentan indole (0.25mM). F - unmodified bacterial cellulose.

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 indigo synthesis did not inhibit the growth of E. coli as seen in Figure 3.

Figure 3: growth burden of pKARA_RT3 composite.

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.