Part:BBa_K3904401

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, team created probiotics capable of naringenin biosynthesis. For the diagnostic part, 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 enditing technique. In our approach to edit E. coli Nissle 1917 genome, we have used two plasmid based system enableing to combine Lambda Red recombination and CRISPR-Cas9 as cunterselection tool - pCas (could be found in addgene: #62225) and pTarget (could be found in addgene as pTargetF: #62226).

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 inducable promoter araBp. pTarget plasmid caries consitutively expressed signle-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 lenght RNA sequence complementary to the targeted DNA adjecent 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 exists as two seperate RNA molecules, however, in laboratory experiments they are usually combined into one single-guide RNA (sgRNA). As both pCas and pTarget plasmids are in cell, Cas9 nuclease and sgRNA are able to form ribonucleoprotein complex and perform double strand break in the chosen part of the DNA. However, if arabinose has been added to the cell culture and double stranded DNA repair tamplet is present in the cell, Lambda Red system performs homologous recombination. If this process is unsuccessful, Cas9-sgRNA complex will cause double strand break and will cause cell death (2). This is employed as a counterselection in order to avoid of the additional antibiotic as selection marker usage.

Plasmid curing pCas also caries sgRNA targeting pTarget plasmid. This sgRNA is expressed under the controle of IPTG inducable lac operon. As IPTG added to the culture media it binds repressor lacI and allows to begin transcription of sgRNA targeted to pTarget replicon. pCas plasmid is cured by growing overnight culture at 37°C.

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
  COMPATIBLE WITH RFC[25]
• 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. Ku, J. T., Chen, A. Y., & Lan, E. I. (2020). Metabolic engineering design strategies for increasing acetyl-CoA flux. Metabolites, 10(4), 166.
4. 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.