Part:BBa_K5246058
Ds - donor DNA for wzyE Gene Impairment
Introduction
Vilnius-Lithuania iGEM 2024 project Synhesion aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - C. crescentus and H. baltica - harness 12 protein synthesis pathways to produce sugars, anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used E. coli bacteria to produce adhesives. Our team concomitantly focused on creating a novel E. coli strain for more efficient production of adhesives.
Usage and Biology
Biology
The CRISPR/Cas9 system is composed of a guide RNA (gRNA) and the endonuclease Cas9. Together, they form a complex that directs the enzyme to the target site complementary to the gRNA, facilitating site-specific cleavage [1]. The enzymatically induced double-strand break (DSB) is subsequently repaired by the cell via either homologous recombination (HR) or non-homologous end joining (NHEJ). However, NHEJ works poorly in E. coli [2]. Most methods for modifying chromosomal DNA in E. coli utilize phage recombinase-mediated homologous recombination, commonly known as recombineering. This can be achieved through the Rac prophage system or the three bacteriophage λ Red proteins: Exo, Beta, and Gam. Including the λ Red machinery significantly enhances mutagenesis efficiency, particularly when used in conjunction with CRISPR/Cas9 technology. The λ Red system requires template DNA to facilitate the repair process [3].
WzyE catalyses polymerization of repeat unit Und-P linked saccharides at the periplasmic face of the inner membrane via a 'block-transfer' mechanism with modulation of polysaccharide chain length dependent on WzzE. [5].
Eliminating the wecA gene results in ECA and lipopolysaccharide 08-side chain inactivation without causing high-stress levels [6].
Usage
Knocking out the wecA gene and inactivating the ECA pathway loads off the metabolic burden of synthesizing polysaccharides. It prevents ECA biosynthesis and increases precursor availability for peptidoglycan biosynthesis [7]. BL21(DE3)∆wecA strain could be used to investigate the ECA pathway further and the cell interactions with other cells or bacteriophages. This strain could be used in biotechnology as cells with altered surface properties. Biofilms are increasingly recognized as a critical global issue in many industries and come out with economic costs. Biofilms contaminate manufacturing equipment and pharmaceuticals, compromising product quality and safety. Biofilms can foul heat exchangers, pipelines, and other industrial equipment, leading to decreased performance and increased energy consumption. A strain with reduced biofilm formation could save power consumption and finances [8]. On top of that, exposed glycan structures often serve as initial receptors for host - N4 bacteriophage - recognition. Subsequent binding to a terminal or secondary receptor directly on the cell surface triggers irreversible adsorption and injection of the phage genome. [9] N4 bacteriophage infects industrially grown bacteria. Eliminating recognition antigens on these bacteria could provide them additional resistance to N4 phage infection. [10].
In our project, Synhesion, we aimed to produce an efficient polysaccharide in E. coli by introducing a new synthesis pathway. This pathway originated from the bacteria C. crescentus</> and <i>H. baltica, which inhabit aquatic environments. Although polysaccharide synthesis pathways in E. coli and marine bacteria are analogous, we aimed to engineer an E. coli strain that could produce desired polysaccharides more efficiently by reducing its metabolic burden. For this reason, we chose to eliminate the polysaccharide-producing ECA pathway from E. coli by knocking out the wecA gene.
dDNA was used as a repair template in Multiple Stepwise Gene Knockout.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Experimental characterization
See Vilnius-Lithuania iGEM 2024 project Synhesion for more information.
References
1.Sander, J. D., & Joung, J. K. (2014). CRISPR-Cas systems for editing, regulating, and targeting genomes. Nature Biotechnology, 32(4), 347–355. https://doi.org/10.1038/nbt.2842
2. Wright, D. G., Castore, R., Shi, R., Mallick, A., Ennis, D. G., & Harrison, L. (2016). Mycobacterium tuberculosis and Mycobacterium marinum non-homologous end-joining proteins can function together to join DNA ends in Escherichia coli. Mutagenesis, 32(2), 245–256. https://doi.org/10.1093/mutage/gev083
3.Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3), 233–239. https://doi.org/10.1038/nbt.2500
4.Rai, A. K., Carr, J. F., Bautista, D. E., Wang, W., & Mitchell, A. M. (2021). ElyC and cyclic enterobacterial common antigen regulate synthesis of phosphoglyceride-linked enterobacterial common antigen. mBio, 12(5), e02846-21. https://doi.org/10.1128/mBio.02846-21
5.Kajimura, J., Rahman, A., & Rick, P. D. (2005). Assembly of cyclic enterobacterial common antigen in Escherichia coli K-12. Journal of Bacteriology, 187(20), 6917–6927. https://doi.org/10.1128/JB.187.20.6917-6927.2005
6.II Fuc4NAc transferase involved in synthesis of enterobacterial common antigen in Escherichia coli K-12. Journal of Bacteriology, 183(22), 6509–6516. https://doi.org/10.1128/JB.183.22.6509-6516.2001
7.Nobrega, F. L., Vlot, M., de Jonge, P. A., Drost, M., & Brouns, S. J. J. (2018). Targeting mechanisms of tailed bacteriophages. Nature Reviews Microbiology, 16(12), 760–773. https://doi.org/10.1038/s41579-018-0070-8
8.McPartland, J., & Rothman-Denes, L. B. (2009). The tail sheath of bacteriophage N4 interacts with the Escherichia coli receptor. Journal of Bacteriology, 191(2), 525–532. https://doi.org/10.1128/JB.01423-08
9.Rick, P. D., Hubbard, G. L., & Barr, K. (1994). Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12. Journal of Bacteriology, 176(9), 2877–2884. https://doi.org/10.1128/jb.176.9.2877-2884.1994
10. Klena, J. D., & Schnaitman, C. A. (1993). Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Molecular Microbiology, 9(2), 393–402. https://doi.org/10.1111/j.1365-2958.1993.tb01705.x
11. Alexander, D. C., & Valvano, M. A. (1994). Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine. Journal of Bacteriology, 176(22), 7079–7084. https://doi.org/10.1128/jb.176.22.7079-7084.1994
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