Improve
The main component of our bioink is alginate, produced by Azotobacter vinelandii. Alginate is a polysaccharide, with a consistency of jelly or gum, which makes it hard to use as a solid base for our printing. To enhance the stability and strength of our printed product, we decided to introduce a second polysaccharide, which is bacterial cellulose. In the following, by cellulose is always bacterial cellulose from the Komagataeibacter xylinus cellulose opereron meant. (bacterial cellulose is a biopolymer enhancing cell-walls from bacteria).
Genetic compounds
Cellulose is not produced by A. vinelandii why we chose to introduce the cellulose operon from Komagataeibacter xylinus, which is one of the most well known bacterial-species for efficient cellulose production 1 . For the cellulose production in K. xylinus 4 enzymes are needed which synthesize and structure the polysaccharide, all those enzymes could be found in the iGEM-registry (see table 1). The main enzyme is the cellulose synthase, which consists of 4 subunits acsA-D 2. To help getting the cellulose-fibers in the right arrangement an endo-beta-1,4-glucanase and cellulase-complementing protein is needed, additionally a β-glucosidase can hydrolyze cellulose fibers, when assembled in the wrong arrangements. It took a long time to find all the sub-parts from the cellulose operon (see table 1) in the registry, so we decided to create a complete cellulose operon BBa_K4369001, with all the proteins in the right order. We also uploaded all sub-parts with the improvements we made to the registry, because the whole operon might be too huge to synthesize it at once.
| Name | Function | Part number | Number of improved part | Improvements |
|---|---|---|---|---|
| acsAB | A: catalytic domain B: regulatory domain | BBa_K1321334 | BBa_K4369004 | His-tag |
| acsCD | cellulose excretion important for optimal production yields | BBa_K1321335 | BBa_K4369005 | His-tag |
| β-glucosidase | hydrolyze tangled glucan fibers when wrong arrangement of the chains | BBa_BBa_K3605006 | BBa_K4369006 | codon optimisation for E. coli, His-tag RBS (ribosomal binding site) |
| endo-beta-1,4-glucanase and cellulase-complementing protein | important for the structure of the cellulose fibers | BBa_K2804010 | BBa_K4369003 | His-tag |
Fortunately all subunits of the cellulose synthase were part of the 2021 distribution kit. Our plan was to combine subunit A-D in one vector to be introduced in A. vinelandii and to evaluate if this is enough to produce cellulose.
With the parts BBa_K1321334, BBa_K1321335 the iGEM14_Imperial team mentioned in their parts description that they achieved small amounts of cellulose. That is why in a further step we planned to also introduce the part BBa_K2804010 (see table 1), as the iGEM18_Ecuador who made this planned to use their part together with the parts for subunit acsAB and acsAC, but weren't successful with this.
As mentioned in table 1, most parts were codon-optimized for E.coli, so we decided as a first step, to optimize the codons for our A. vinelandii. But when designing the gene constructs we faced the problem, that this codon optimization for A. vinelandii introduced so many guanines and cytosines in the genes, that a synthetisation was not possible. We did notfind a solution to this problem and decided to test the cellulose production in E. coli first, as we knew from the registry it would definitely work there. We chose the pET21 vector with an ampicillin resistance as the backbone and added a His-tag behind every subunit gene to be able to detect them with an affinity protein purification.
Optogenetic inducement
Our overall goal was to introduce all enzymes that are needed for a successful cellulose production intro A. vinelandii. In the workflow of our printing process, the start of cellulose exudation must be timed correctly, as too early exudation can clog the needle and too late exudation can lead to dispersion of the ink. To start the cellulose production we planned to use the optogenetic induced pDawn system, which is promoted by blue light 3 . The optogenetic part of the pDawn system is the light-oxygen-voltage (LOV) domain, which is induced by blue light. If blue light activates this domain a protein-cascade leads to the transcription of the connected genes of interest, which is here the cellulose-operon.
Conclusion
Due to the problem of codon optimization and the fact that A. vinelandii needs a genomic integration of new genes,4 we were not able to perform the introduction of the cellulose operon to the pDawm system and A. vinelandii as planned. Still we were able to improve the existing iGEM parts BBa_K1321334, and BBa_K1321335 by adding His-tags to them and we also did the in silico cloning of the complete cellulose operon BBa_K4369001 containing all the proteins which are needed for a complete cellulose operon.
We think combining cellulose expression with optogenetic switches is a new attempt in iGEM, also we prepared a good base to perform this step in A. vinelandii and E. coli, which is a good benefit for other teams.
- Shin-Ping Lin, 2013, Biosynthesis, production and applications of bacterial cellulose DOI: 10.1007/s10570-013-9994-3
- M. Liu et al, 2018, Complete genome analysis of *Gluconacetobacter xylinus* CGMCC 2955 for elucidating bacterial cellulose biosynthesis and metabolic regulation, DOI: 10.1038/s41598-018-24559-w
- R. Ohlendorf et al, 2012, From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression, doi: 10.1016/j.jmb.2012.01.001
- Ribbe, Markus W. (2011). [Methods in Molecular Biology] Nitrogen Fixation Volume 766 || Molecular Biology and Genetic Engineering in Nitrogen Fixation. , 10.1007/978-1-61779-194-9(Chapter 6), 81–92. doi:10.1007/978-1-61779-194-9_6