pHimarEm1

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 6663
Illegal NheI site found at 330
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 6669
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 6663
Illegal BglII site found at 652
Illegal BglII site found at 3191
Illegal BglII site found at 3850
Illegal XhoI site found at 287
Illegal XhoI site found at 3488
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found at 6663
Illegal suffix found at 2
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found at 6663
Plasmid lacks a suffix.
Illegal XbaI site found at 6678
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NgoMIV site found at 1316
Illegal AgeI site found at 2883
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.

This is the sequence for the backbone of one of the main plasmids that is used in order to genetically manipulate several categories of γ-proteobacteria, including Flavobacteriia\cite{mcbride1996}.

Description

This rather big plasmid has long been used in order to integrate genetic material into the genome of Flavobacteria\cite{someotherthing}. While it is 6 kb long, not all of them are functional. The main points of interest of this plasmid are its gene encoding a protein that confers resistance to Kanamycin and Neomycin (KanR from Tn4351), Aminoglycosidase phosphotransferase and the gene encoding a protein that confers resistance to Erythromycin (ErmF from Bacteroides fragilis), rRNA adenine N-6-methyltransferase.
The interesting fact about this plasmid is that the KanR gene is expressed in most E. coli strains, whereas ErmF is not\cite{somethirdthing}. In stark contrast, the ErmF gene is expressed in other bacteria, such as Flavobacteriia, making pHimarEm1 a prime candidate for conjugation based genetic transfer, especially for bacteria where transformation is not as efficient as conjugation. The precise reason that ErmF is not expressed in most E. coli is not known, to our knowledge.
Furthermore, the plasmid carries the Mariner transposase\cite{somemorestuff} and the corresponding inverted repeats (IRs), which are: GGGGGGGGGGGGGGGG and CCCCCCCCCCCCCCCCCC.
In order to make transposition inducible, the transposase is placed under the control of an inducible lac promoter using the regular lac operon\cite{somefifthstuff}. This means that transposition can be initiated at will by introducing IPTG to the growth medium.
Finally, the R6K γ ori ensures that the plasmid can be stably replicated when not integrated in the bacterial chromosome.

Optimization & Protein Analysis

BcsD seems to be required for the arrangement of the BCS complex along the longitudinal cell axis. Although most biofilm-forming bacteria likely produce amorphous cellulose that is embedded in a 3D matrix of polysaccharides, proteinaceous fibers, and nucleic acids, some bacteria produce cellulose microfibrils resembling those synthesized by eukaryotic cells. In such bacteria, CesA complexes are linearly arranged along the cell axis, and the CesA operons encode at least one additional subunit, BcsD, that might facilitate the linear organization of the synthases.

Athens 2020

The current part is utilised by the iGEM Athens 2020 team during the project MORPHÆ. In this project, Flavobacteria were used to produce a non-cellular structurally coloured biomaterial which would require the secretion of a biomolecule that Flavobacteria do not normally secrete. Our hypothesis is that the formed matrix will have a structure similar to that of the biofilm and thus, it will provide the material with macroscopically the same colouration properties as the biofilm. In order to transfer the desired genes into the genome of Flavobacterium johnsoniae, our chassis of choice, we utilise conjugation of the strain UW101 with E. coli S17-1. Once conjugation is complete, transposition will be induced, in order to integrate the genes of interest into F. johnsoniae's chromosome, after which expression will occur.

SOURCE OF THIS PART

The nucleotide sequences of the bacterial cellulose operon come from the strain Komagataeibacter xylinus and GenBank database (Acc.No.X54676.1). K.xylinus is a member of the acetic acid bacteria, a group of Gram-negative aerobic bacteria that produce acetic acid during fermentation.

Useful Links:

NCBI taxonomy:

https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=28448&lvl=3&lin=f&keep=1&srchmode=1&unlock

GenBank link:

https://www.ncbi.nlm.nih.gov/nuccore/X54676.1

Codon optimisation bank:

http://genomes.urv.es/OPTIMIZER/?fbclid=IwAR0ALbP_C8UVY4itvYdNX8b5KYYUM5ulQojz8UJAK6Zj5llobNNxE-jYmXQ

Codon optimization table:

https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=376686&fbclid=IwAR0gwwrIarZsiYhWvHPc2BKy-iB_2OM-DPB5I2HYJZwBNiasmlLXWK87PwM

REFERENCES

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

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

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

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

Nakamura, Y. (2000). Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Research, 28(1), 292-292. doi: 10.1093/nar/28.1.292

Omadjela, O., Narahari, A., Strumillo, J., Melida, H., Mazur, O., Bulone, V., & Zimmer, J. (2013). BcsA and BcsB form the catalytically active core of bacterial cellulose synthase sufficient for in vitro cellulose synthesis. Proceedings Of The National Academy Of Sciences, 110(44), 17856-17861. doi: 10.1073/pnas.1314063110