Part:BBa_K3904403

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.

adhE gene knockout

This 20 nt length RNA sequence is designed to target acetaldehyde dehydrogenase (adhE) gene. Knockout of adhE gene disrupts the conversion of acetyl-CoA to ethanol in the cell, therefore more acetyl-CoA can be converted to malonyl-CoA (3).

Importance to AmeBye project

Malonyl-CoA is used by chalcone synthase during the production of naringenin chalcone, which is later converted to naringenin (3).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 9
  
• 1000
  COMPATIBLE WITH RFC[1000]

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. Wu, J., Du, G., Chen, J., & Zhou, J. (2015). Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli. Scientific reports, 5(1), 1-14.