Revision as of 19:55, 20 September 2021

Introduction

Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefor target E. histolytica from several angles: prevention and diagnostics. As a tool to prevent amebiasis, the team created probiotics capable of naringenin biosynthesis. For the diagnostic part, the project includes a rapid, point of care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers, specific to the E. histolytica secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.

Usage and Biology

CRISPR-Cas9 is a versatile genome-editing technique. In our approach to editing E. coli Nissle 1917 genome, we have used two plasmid based system enabling to combine of Lambda Red recombination and CRISPR-Cas9 as counterselection tools - pCas and pTarget.

Mechanism of genome editing

pCas plasmid is used for Cas9, Lambda Red system expression, and plasmid curing of pTarget. Cas9 - the RNA-guided endonuclease - is expressed constitutively, while the expression of Lambda Red genes (Gam, Exo, Beta) is under the control of arabinose inducible promoter araBp. pTarget plasmid caries constitutively expressed single-guide RNA (sgRNA). This RNA molecule, as and in nature, is composed of two central parts: CRISPR RNA (crRNA) and tracrRNA. crRNA is 17-20 nt length RNA sequence complementary to the targeted DNA adjacent to the protospacer adjacent motif (PAM) and tracrRNA is the scaffold for the Cas (in this case Cas9) nuclease binding to guide RNA and forming the ribonucleoprotein complex (1). In nature those two parts exist as two separate RNA molecules, however, in laboratory experiments they are usually combined into one single-guide RNA (sgRNA). As both pCas and pTarget plasmids are in a cell, Cas9 nuclease and sgRNA are able to form ribonucleoprotein complex and perform a double-strand break in the chosen part of the DNA. However, if arabinose has been added to the cell culture and a double-stranded DNA repair template is present in the cell, the Lambda Red system performs homologous recombination. If this process is unsuccessful, the Cas9-sgRNA complex will cause a double-strand break and will cause cell death (2). This is employed as a counterselection in order to avoid the additional antibiotic as selection marker usage.

tyrP gene knockout

This 20 nt lenght RNA sequence is designed to target gene. tyrP gene is known to be involved in transporting tyrosine across the cytoplasmic membrane, therefore tyrP knockout mutants are potentially able to produce 10 % higher amounts of L-tyrosine than that of the original strains (3).

Importance to AmeBye project

This L-tyrosine then can be converted to p-coumaric acid by tyrosine ammonia-lyase (TAL), which is further utilized to make p-coumaroyl-CoA by 4-coumarate: CoA ligase. The sequential condensation of one p-coumaroyl-CoA and three malonyl-CoA leads to the formation of naringenin chalcone by chalcone synthase (CHS), which is then converted to naringenin by chalcone isomerase (CHI) (4).

Sequence and Features

References

1. Doudna, J. A., & Charpentier, E. (2014). The new frontier of genome engineering with CRISPR-Cas9. Science, 346(6213).
2. Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., & Yang, S. (2015). Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Applied and environmental microbiology, 81(7), 2506-2514.
3. Wang, Q., Zeng, W., & Zhou, J. (2019). Effect of gene knockout of L-tyrosine transport system on L-tyrosine production in Escherichia coli. Sheng wu gong cheng xue bao= Chinese journal of biotechnology, 35(7), 1247-1255.
4. Ganesan, V., Li, Z., Wang, X., & Zhang, H. (2017). Heterologous biosynthesis of natural product naringenin by co-culture engineering. Synthetic and systems biotechnology, 2(3), 236-242.