Part:BBa_K3520006:Design

ccpAx-gene for Bacterial Cellulose upregulation

DESIGN CONSIDERATIONS

1. Codon optimization for Flavobacteria johnsoniae UW101.

As a first step, all the sequences of the genetic construct were codon-optimized for the increased expression in Flavobacterium johnsoniae UW101, according to the Codon Usage Table UW101 (Nakamura, 2000). This specific table was chosen for two main reasons. Firstly, it is the most thoroughly documented one, featuring the biggest CDS count out of many closely related taxa. Secondly, as these CDSs will be used in Flavobacteria johnsoniae, Flavobacteria IR1, and Flavobacteria IR1 M16, the codon optimisation should be the closest in all three. After reviewing the phylogeny of the genus, and taking into consideration the closest known relatives of Flavobacteria IR1, i.e. Flavobacterium aquidurense and Flavobacterium pectinovorum, we started looking at the different representations of codons in the genomes of various Flavobacteriia. We saw that the differences were negligible among the documented species. This led us to use the most represented item in the database.

2. Elimination of restriction sites for illegal sites of TYPE IIS and RFC10 Assembly

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, an RBS, and a terminator. Specific prefixes and suffixes are required in order to isolate and assemble the parts. The parts are flanked by fusion sites that ensure assembly in the right order, and a BsaI restriction enzyme site. RFC 10 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 each 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.

3. Primers design

Internal primers for PCR amplification prior to level 0 Golden Gate assembly. Similarly, internal primers were placed between the SapI sites and transcriptional units, in order to perform further amplification prior to performing the level 1 Golden Gate assembly. The primary reason that this was done is to increase the chances of Golden Gate assembly functioning, as the parts that would be ligated, especially at the level 1 stage, are rather large. The RFC10 prefix and suffix already have well-established primers (VW and VW-R) that most iGEM members are familiar with, forgoing the need to order very different primers to amplify each part. thus reducing cost.

We hope all of the above will make it easier for future teams that work with the particular species and gives them higher manipulation capabilities and accuracy.

References

Villads Egede Johansen, Laura Catón, Raditijo Hamidjaja, Els Oosterink, Bodo D. Wilts, Torben Sølbeck Rasmussen, Michael Mario Sherlock, Colin J. Ingham, Silvia Vignolini, Genetic manipulation of structural color in bacterial colonies, PNAS March 13, 2018 115 (11) 2652-2657; first published February 22, 2018; https://doi.org/10.1073/pnas.1716214115

Max Kolton, Noa Sela, Yigal Elad, and Eddie Cytryn, Comparative Genomic Analysis Indicates that Niche Adaptation of Terrestrial Flavobacteria Is Strongly Linked to Plant Glycan Metabolism, PLoS One. 2013; 8(9): e76704. Published online 2013 Sep 26. doi: 10.1371/journal.pone.0076704

Chen, S., Bagdasarian, M., Kaufman, M., & Walker, E. (2006). Characterization of Strong Promoters from an Environmental Flavobacterium hibernum Strain by Using a Green Fluorescent Protein-Based Reporter System. Applied And Environmental Microbiology, 73(4), 1089-1100. doi: 10.1128/aem.01577-06

Chen, S., Kaufman, M., Bagdasarian, M., Bates, A., & Walker, E. (2010). Development of an efficient expression system for Flavobacterium strains. Gene, 458(1-2), 1-10. doi: 10.1016/j.gene.2010.02.006