Team:Athens/Design
DESIGN
The inspiration for our project came from observing nature and its unique mechanisms for producing colouration. More specifically, structural colour, which is a result of periodic structures in the microscale and their interaction with light, is found in animals, plants and some strains of bacteria [1]. Through following the principles of Biomimicry, these natural structures could be applied to any material, in order to provide the same effect of structural colouration. Thus, the aim of our project is the creation of a biomaterial that can have the appropriate structure, so as to appear coloured and that is safe, biodegradable and sustainable to produce.
After reviewing the existing literature on the formation of structural colour in bacteria, we came across Flavobacterium johnsoniae and the Flavobacterium IR1 strain. These species are naturally structurally coloured when forming a biofilm, as a result of the defined spatial arrangement of the cells. Research done thus far suggests that structural colouration in Flavobacteriia is related to gliding motility, a novel mechanism of cellular translocation, that does not involve flangellae or pilli, and is mediated by the T9 secretion system (T9SS), a novel bacterial secretory pathway, but the mechanisms and cause of its formation still remain unknown [2].
Initially, we were considering transferring this system to E. coli, in order to manipulate their spatial arrangement in a manner which would result in structural colouration similar to the Flavobacteriia. However, after contacting Dr. Colin Ingham, we concluded that the transfer of the system would require extensive engineering with uncertain success, mainly due to the lack of knowledge underlying the formation of structural colour. Instead, we decided to genetically manipulate Flavobacteriia to form the desirable biomaterial. The bacteria we intend to use have already shown intense structural colour [2]. These are Flavobacterium johnsoniae, IR1 wt and IR1 M16 and they were provided to us by Dr. Ingham along with the appropriate plasmids and E. coli S17-1, a strain widely used for bacterial conjugation, a strain that has shown widely used for bacterial conjugation. [3]. According to the literature, the mutant M16 exhibits the most intense structural colouration, but we intend to conduct experiments using all of the strains of bacteria given to us in order to test and study the interaction between each strain of bacteria and cellulose [2].
The use of Flavobacteriia to produce a cell free, structurally coloured biomaterial would require the secretion of a biomolecule that Flavobacteriia do not normally secrete. Our hypothesis is that the matrix that will be formed will have a structure similar to that of the biofilm which created it and thus, it will provide the material with macroscopically the same colouration properties as the biofilm. Our initial idea was to use bacterial cellulose as an appropriate biomaterial, because of its unique properties, robustness and biodegradability [4].
The genes responsible for its production were selected from the bcs operon of Komagataeibacter xylinus (Acc. No. X54676.1), the most efficient bacterial cellulose producer, which consists of four genes, bcsA, bcsB, bcsC and bcsD [5]. bcsA is responsible for the encoding of the catalytic subunit and bcsB of the regulatory subunit of the cellulose synthase. The function of bcsC and bcsD still remains undetermined, but it has been suggested that bcsC is responsible for the creation of pores in the membrane, while bcsD is involved in the crystallization of cellulose into nanofibrils. Furthermore, we found that the incorporation of the upstream genes, cmcax and ccpAx, would also be meaningful in multiplying the production of cellulose [6].
In order for the genes to be inserted in the Flavobacteriia, they will be firstly incorporated into an appropriate vector. These bacteria, however, do not have the ability to be transformed using conventional vectors, because they tend to reject the inserted plasmid [7]. Therefore, we could utilize a transposase for the insertion of our genetic construct directly in the genome of the recipient Flavobacterium, without the need of a replicative plasmid. After further communication with Dr. Colin Ingham, he provided us pHimarEm1, a plasmid which harbors the mariner transposase Himar1. This plasmid was originally designed to be used in identifying the gliding motility genes of Flavobacterium johnsoniae by mariner mutagenesis. The random targeting of the transposon in the genome has the potential to knock out genes, and those null mutants are in turn analysed for their phenotype. This system could be used to our advantage, by changing the transposed sequence, from one which contains just a selection marker, to one which also contains our construct. The plasmid backbone also contains two different antibiotic resistance genes, to erythromycin and kanamycin, to confer resistance to the final Flavobacterium strain, and to the E. coli intermediate, respectively. [2].
In order for the genes to be successfully inserted into the plasmid, and to be made accessible to the whole of the iGEM community, they will firstly be made into BioBricks. This will be done according to the Type IIS Assembly protocol, but the parts will be compatible with both the RFC10 and Type IIS standards. The latter protocol was chosen because of its precision as
it eliminates scarring between connected modules, or CDSs. This was vital to our design, as the CDSs constituting our construct exceeded the upper limit of synthesizable DNA, provided by iGEM sponsors, IDT and Twist Bioscience. The RBS was chosen based on its compatibility and performance in Flavobacteriia, according to existing literature [8]. The promoter we intend on using is the opmA promoter, a strong constitutive promoter found in a well characterised strain of Flavobacterium johnsoniae [9]. Lastly, the terminator was chosen from the existing iGEM parts (BBa_B0015), due to its high efficiency and compatibility with RCF10 and Type IIS.
All the sequences of the genetic construct were codon optimized for the increased expression in Flavobacterium 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 Flavobacterium johnsoniae, Flavobacterium IR1, and Flavobacterium 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 FlavobacteriumIR1, 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 in the database. Furthermore, all the illegal sites for the restriction enzymes of the protocols we plan on following are eliminated. Lastly, appropriate prefixes and suffixes are chosen for each module, in order for the formation of a transcriptional unit for each gene, that will be then inserted into the plasmid.
Each level 0 module that the BioBrick will contain (promoter, CDS, RBS, terminator) is flanked by a BsaI site as well as a special four nucleotide sequence, unique for each part. The 4N sequence is chosen in order to connect each part with the next in the correct order in a single pot reaction. Therefore, once each module is digested and ligated, a transcriptional unit will form containing a promoter, RBS, CDS and a terminator [10].
Similarly to the ligation of the level 0 modules, each resulting transcriptional unit will be flanked by the SapI site as well as a 3N special sequence. Additionally for every part, the prefix and suffix of the RFC10 protocol will be inserted. Again, in a single pot reaction the different TUs will be digested with SapI and ligated. The multi-transcriptional unit that forms will contain an appropriate prefix and suffix, in order to connect with the pHimarEm1 plasmid backbone [10].
All in all, by choosing this cloning method and simultaneously incorporating the constructs into biobricks, the RFC10 assembly standard’s prefixes and suffixes, the construction becomes easily reproducible and extendible to different systems and organisms.
In order to successfully insert the non replicating plasmid harboring the transposase and our MultiTranscriptional Unit into the Flavobacteriia, we chose conjugation as a prefered method. The use of conventional methods, such as electroporation or heat shock, have been proven less successful according to the existing literature, contrary to conjugation [7]. Firstly, the plasmid will be inserted into competent E. coli S17-1 bacteria, and only the recombinant bacteria will be isolated via antibiotic selection, using kanamycin. These will in turn be co-cultivated with Flavobacteriia in order for conjugation to occur. After a successful conjugation, the bacteria are plated on a plate containing erythromycin, which permits growth for only the recombinant Flavobacteriia to survive, and kills off all E. coli and non recombinant Flavobacteriia.
The successful incorporation of the transposon into the genome of the Flavobacterium will be checked with classical molecular biology techniques. By selecting the right primers, a PCR reaction will show the existence of our construct inside the genome. This incorporation is also suspected to result in phenotypic differentiation. As remarked by Dr. Ingham, the expression of the cellulose synthesis genes, and the subsequent cellulose production will give rise to a more rigid extracellular matrix and the spatial relations between neighbouring cells are expected to be slightly disrupted, giving rise to a macroscopically observable change in colouration.
After the production of cellulose at the extracellular space of the bacteria, the next step for our project is the isolation of the cellulose matrix. In order to distinguish the final product from living cells, two upcoming steps will be performed. We separate BC films from the cell culture and the separated product will be dried as described in existing literature, following the room temperature drying method [11]. More specifically, the BC films are harvested, immersed in ethanol, transferred to deionized water and boiled multiple times within 24 h. This method will result in the final product: a cellulose film without any Colony Forming Units, and able to retain the microscopic structure which gives rise to its structural colour.
To estimate specific properties of our biomaterial, we designed a set of models that can be found on the Background page.
[1] Sun, J., Bhushan, B., & Tong, J. (2013). Structural coloration in nature. RSC Advances, 3(35), 14862. doi: 10.1039/c3ra41096j
[2] Braun, T., Khubbar, M., Saffarini, D., & McBride, M. (2005). Flavobacterium johnsoniae Gliding Motility Genes Identified by mariner Mutagenesis. Journal Of Bacteriology, 187(20), 6943-6952. doi: 10.1128/jb.187.20.6943-6952.2005
[3] Strand, T., Lale, R., Degnes, K., Lando, M., & Valla, S. (2014). A New and Improved Host-Independent Plasmid System for RK2-Based Conjugal Transfer. Plos ONE, 9(3), e90372. doi: 10.1371/journal.pone.0090372
[4] Wang, J., Tavakoli, J., & Tang, Y. (2019). Bacterial cellulose production, properties and applications with different culture methods – A review. Carbohydrate Polymers, 219, 63–76.
[5] Johansen, V., Catón, L., Hamidjaja, R., Oosterink, E., Wilts, B., & Rasmussen, T. et al. (2018). Genetic manipulation of structural color in bacterial colonies. Proceedings Of The National Academy Of Sciences, 115(11), 2652-2657. doi: 10.1073/pnas.1716214115
[6] Buldum, G., Bismarck, A., & Mantalaris, A. (2017). Recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli. Bioprocess And Biosystems Engineering, 41(2), 265-279. doi: 10.1007/s00449-017-1864-1
[7] McBride, M., & Kempf, M. (1996). Development of techniques for the genetic manipulation of the gliding bacterium Cytophaga johnsonae. Journal Of Bacteriology, 178(3), 583-590. doi: 10.1128/jb.178.3.583-590.1996
[8] 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
[9] 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
[10] Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. Plos ONE, 6(2), e16765. doi: 10.1371/journal.pone.0016765
[11] Zeng, M., Laromaine, A., & Roig, A. (2014). Bacterial cellulose films: influence of bacterial strain and drying route on film properties. Cellulose, 21(6), 4455-4469. doi: 10.1007/s10570-014-0408-y
"Papilio ulysses butterfly" by Deanster1983 who's mostly off is licensed under CC BY-ND 2.0
