Team:Athens/Contribution
CONTRIBUTION
Our team provides new insight and information about the Flavobacterium genus. Most importantly, we created a de novo model of bacterial mobility and we provide an easy-to-follow process to add new genes to bacteria of this genus. This certain process can be applied to any gene or circuit of genes that researchers desire to express in Flavobacteriia species. The genes we used in this project are involved in the production and extracellular secretion of cellulose, but the same process can be applied to other genes.
In order to produce cellulose in Flavobacterium johnsoniae, we used the genes responsible for cellulose production from Komagataeibacter xylinus. Some of these genes were part of the bcs operon and are bcsA, bcsB, bcsC and bcsD (Acs.No. X54676.1), while others are found upstream of the operon and are related to the regulation of the previous ones like cmcax ccpAx. The Type IIS Assembly standard was used in order to insert these genes in the pHimarEm1 plasmid, avoiding the presence of illegal sites.
Each gene will be assembled with a promoter, RBS and a terminator [1], [2]. Specific prefixes and suffixes are required in order to isolate and assemble the parts. The parts are flanked by fusion sites that ensure proper order assembly and a BsaI restriction enzyme site. RFC10 assembly standard-compatible BioBrick prefix and suffix sequences were added in the 5’ and 3’ ends to allow for easy amplification of the ordered parts as well as sequencing. The synthesised transcriptional unit will consist of the assembled parts, the fusion site 5’ of the promoter, and the fusion site 3’ of the terminator.
Once the transcriptional unit of each gene is synthesised, they will be inserted in the pHimarEm1 plasmid in one step using type IIS assembly, in the designated order due to the 5’ and 3’ fusion sites.
For that procedure we either created new parts and added them to the registry or modified already existing parts to increase their expression to the Flavobacterium genus, utilising codon optimisation. We hope all of the above will make it easier for future teams to adopt the particular strategies and parts so it becomes easier to work with these species and allow for more precise genetic manipulation.
Basic Parts
| Part Name | Short Description | Part Type | Length (bp) |
|---|---|---|---|
| BBa_K3520031 | bcsA-Bacterial Cellulose Synthase A | Coding | 2172 |
| BBa_K3520002 | bcsB-Regulatory Subunit of Bacterial Cellulose operon | Coding | 2483 |
| BBa_K3520003 | bcsC- part of Bacterial Cellulose operon | Coding | 3909 |
| BBa_K3520004 | bcsD-part of Bacterial Cellulose operon | Coding | 478 |
| BBa_K3520005 | cmcax: gene for Bacterial Cellulose upregulation | Coding | 1029 |
| BBa_K3520006 | ccpAx: gene for Bacterial Cellulose upregulation | Coding | 1062 |
| BBa_K3520007 | ompA promoter for Flavobacteriia | Regulatory element | 90 |
| BBa_K3520008 | Flavobacteriia RBS | RBS | 5 |
| BBa_K3520009 | pHimarEm1 | Plasmid backbone | 6656 |
| BBa_K3520010 | GFP superfolder for Flavobacteriia | Coding | 717 |
| BBa_K3520011 | Reflectin for Flavobacteriia | Coding | 1062 |
| BBa_K3520012 | Signal Peptide for translocation to membrane in Flavobacteriia | Coding | 90 |
Composite Parts
| Part Name | Short Description | Part Type | Length (bp) |
|---|---|---|---|
| BBa_K3520013 | Flavobacteriia compatible BcsA transcriptional unit | Transcriptional Unit | 2370 |
| BBa_K3520014 | Flavobacteriia compatible BcsB transcriptional unit | Transcriptional Unit | 2625 |
| BBa_K3520015 | Flavobacteriia compatible BcsC transcriptional unit | Transcriptional Unit | 4106 |
| BBa_K3520016 | Flavobacteriia compatible BcsD transcriptional unit | Transcriptional Unit | 665 |
| BBa K3520017 | Flavobacteria compatible Reflectin transcriptional unit with Signal Peptide | Transcriptional Unit | 1341 |
| BBa_K3520018 | Recombinant pHimarEm1 with Signal peptide/Reflectin TU and GFP TU | Plasmid | 8903 |
| BBa_K3520019 | pMORPHÆ:recombinant pHimarEm1with the insert of BcsABCD transcriptional units | Plasmid | 16450 |
| BBa_K3520020 | TU: GFP | Transcriptional Unit | 909 |
| BBa_K3520021 | Flavobacteriia compatible cmcAx transcriptional unit | Transcriptional Unit | 2369 |
| BBa_K3520022 | Flavobacteriia compatible cpAx transcriptional unit | Transcriptional Unit | 1237 |
As stated in the Model section, an extended version of an existing mass-spring model was used in order to simulate the spatial and temporal coordinates of the flavobacteria. The original model, proposed by Janulevicius et al.[1], provides a mechanical insight of the movement and interactive forces of Myxococcus xanthus, a rod-shaped bacterium famous for its ability to create complex macroscopic structures.
The first step into building this extension was to analyse and fully comprehend the dynamics of the original model. This step essentially led to the development of a MATLAB code which simulates the M. xanthus movement, using the original Janulevicius model.
The development of a computationally stable simulation of an abstract mathematical model can be a hard and time-consuming process, especially for undergraduate students participating in the iGEM competition. Moreover, mass-spring simulations are not taught in most universities (in contrast to computational fluid dynamics for example), and thus the challenge is even harder.
Due to the importance of M. xanthus (and other similar bacteria) in research and technology, it is likely that a future iGEM team will need to simulate their movement using a model that is accepted by the scientific community. The developed scriptis available in the Mathworks file exchange site, along with the extended version proposed by our team. It is fairly easy to use and it is highly customisable (e.g. one can implement specific forces). It is also worth noting that the code will be used in the second phase of our project, and thus will be developed even more.
"Morpho butterfly wing scales" by Carolina Biological Supply Company is licensed under CC BY-NC-ND 2.0
