Difference between revisions of "Part:BBa K4719019"

Revision as of 21:22, 8 October 2023

CBD-ProThr box-AnCDA chitin deacetylase and cellulose binding domain fusion protein
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 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 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.

Bacterial cellulose-chitin polymer was achieved by increasing the production of UDP-N-acetylglucosamine, which can be recognized as a viable substrate for cellulose synthase and incorporated in the bacterial cellulose polymer. We employed two strategies to produce this material. The first approach was to add N-acetylglucosamine into the growth medium BBa_K4719013, and the second one was the production of N-acetylglucosamine by K. xylinus from simple sugars such as glucose, fructose, and saccharose in the growth medium BBa_K4719014. After achieving bacterial cellulose-chitin copolymer, we had to deacetylase this material to produce bacterial cellulose-chitosan copolymer.

Usage and biology

This construct contains a fused cellulose binding domain connected to a linker to the N-terminus of deacetylase AnCDA BBa_K4719011 to ensure a higher degree of deacetylation. For protein purification, 6x his-tag was added to the N-terminus of the cellulose binding domain. The composite is contained in pBAD/HisB plasmid. For this part to be functional in your bacterial cellulose-chitosan copolymer production system, we had to purify recombinant protein coded by this composite.

Experimental characterization

Optimisation of recombinant protein expression

Enzymatic activity

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 recombinant deacetylase CBD-ProThr box-AnCDA did not inhibit the growth of E. coli as seen in Figure 1.