Contribution

Introduction

Throughout this year's competition we worked on providing future iGEM teams with tools for many different applications. Since we worked with three different organisms (Escherichia coli, Saccharomyces cerevisiae and Komagataeibacter xylinus) we developed standardized protocols to collect comparable and valuable data. For this we also built a machine to directly perform mechanical property tests without specialized gear. Furthermore we designed genetic constructs for K.xylinus to enable future teams an easier handling of non-model organisms for related applications. Conveniently, we designed parts that interact with cellulose that is also naturally produced by K.xylinus. Furthermore, we fixed a preexisting part (the OYC-eforRed-dropout, eforRed selection cassette for level 1 vector ; BBa_J435281) from the part registry that had the wrong cloning sites and therefore was not usable for the declared purpose of the part. For achieving the suitable color by mixing of different chromoproteins, we developed a software, which predicts the color accurately for a modular coloring system. Lastly, we constructed a xyloglucan specific binding domain.

Contributions we made:

Fixing the OYC Backbone (BBa_J435281):

When we received our distribution kit, we were planning to use the part BBa_J435281, which is a Open Yeast Collection backbone. Sadly we found that the overhangs after Bsa1 digest are not compatible with regular MoClo Level 1 assembly. Therefore we reported the error and progressed by fixing the part part in silico and then in vitro. After repeated sequencing confirmed the problem, we fixed it with running two consecutive Golden Gate reactions, in which the part was fixed at two locations. To achieve this we used primers with overlapping overhangs, different from the original sequence, in the later desired sequences for level one assembly. We now registerd this fixed part as BBa_K5146045

Property testing machine

Another contribution for future teams is the creation of a simplified, low budget property testing machine (Picture 1). With this, iGEM teams who lack the necessary funding or contacts can do their own mechanical testing on textiles, including tear tests and tensile strength tests.

_{Picture 1: Property testing machine mark 2 (“DAS MASCHIN”) in tear test configuration (left) and tensile strength configuration (right).}

For precise replication of our work, we created a step-by-step building instruction for the machine. Additionally to the assembling of the machine, we provide a self-established protocol to allow teams the use of our tear test [crosslink to simplified tear test protocol].

Lastly, as it is crucial for a functioning property testing machine, we wrote some code for the machine to be used in tear tests.

Genetic constructs (Dye)

One of our contributions for future teams are the constructs designed by the modular dyeing subgroup from our team. They feature: BBa_K5146000 a basic part whose purpose is to connect the cds (in our case chromoproteins) to a cellulose binding domain via a flexible linker and to secrete this construct due to the attached Saccharomyces cerevisiae mating factor and BBa_K5146020 another basic part which fuses two preexisting biobrick parts from the iGEM part registry (BBa_J435350 an inducible promoter induced by IPTG and the RBS BBa_K2680529) the aim of this construct is first of all to effortlessly use a 5` UTR in your MoClo reaction and to lower the chance that the RBS is cutted in E. coli naturally, due to its hairpin structure.

Construct BBa_K5146000

_{in violet the flexible linker, in cyan the cellulose binding domain and in orange the secretion tag ; Alphafold Seed: 1998477677 ; ipTM = - pTM = 0.62}

Inducible K.xylinus strain

We accomplished to create a K.xylinus strain with a knockout that strongly decreases the bacterial cellulose production with these parts (hyper link), as well as replacing the wild type constitutive promoter of the bacterial cellulose operon with an arabine induced promoter with these parts, this allows for the bacterial cellulose production to be regulated.

Model

For future teams to get a deeper understanding of the metabolism of K.xylinus we accomplished a metabolic model where we focused on the carbon uptake of K.xylinus. Furthermore we also conducted an LCA to evaluate the cost efficiency of bacterial cellulose.

Dye Software

To give future teams the option to unlock the colourful properties of chromoproteins we created a software that accurately predicts the mixing of sfGFP, mScarlet and aeBlue to establish a rudimentary CMY (cyan, yellow and magenta) system [to the Software] ; [to our software page for an explanation].

Sticky yeast

We genetically engineered S.cerevisiae to express anchor-proteins to the membrane surface with a cellulose binding domain attached. This allows pinpointing where metabolites are secreted indirectly, by showing where the yeast is present.

Standardized protocols

To ensure steady results we created these Custom Protocols for classic microbiology practices. We share them to provide future teams with a headstart in the laboratory and spare them of the mistakes we made.