Part:BBa_K1773022

Cascade with pLux/cI right promoter and strong RBS

I-F type Cascade complex of I-F type CRISPR-Cas system. The Cascade complex distinguishes target DNA and recruits Cas3 (BBa_K1773003) protein, which degrades the target DNA. Cascade complex is comprised of four proteins: Csy1, Csy2, Csy3 and Cas6f. crRNA region coding crRNAs is necessary for complete Cascade complex.

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 NgoMIV site found at 1891
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2408
  

Usage and Biology

Cascade (CRISPR Associated Complex for Antiviral Defence) complex is an essential component of I-type CRISPR-Cas systems, because it distinguishes foreign DNA and promotes its degradation. This ribonucleoprotein complex is coded by operon of 4 genes: Csy1, Csy2, Csy3 and Cas6f, with stoichiometry of 1:1:6:1 respectively. Also it has a crRNA molecule, which acts like a guide, that brings the whole complex to the target dsDNA sequence. Each Cascade complex has a unique Protospacer Adjacent Motive (PAM) recognition site. The recognizable PAM sequence of this specific I-F Cascade complex from Aggregatibacter actinomycetemcomitans recognizes CC nucleotides directly upstream of the protospacer (the target sequence). In other words, a target sequence needs to have a CC pair on the non-complementary strand of DNA -2 -1 positions of the protospacer. When Cascade complex finds and recognizes the DNA target, then Cas3 (BBa_K1773003) is recruited for DNA degradation.

Expression analysis

Note: This experiment was also done with BBa_K1773023; BBa_K1773024 and they are present in the same immunoblot.

We wanted to test if this Cascade complex part is expressed in E. coli, but the coding cassette of Cascade genes did not have any selectable tag for immunobloting. For that reason we co-transformed another plasmid containing Csy3 gene with a His6 tag along with Cascade biobrick to BL21-DE3 E. coli strain. When Cascade complex gets assembled in the cell, the Csy3 protein with a His6 tag also gets assembled inside the complex. If Cascade complex is expressed - we should see that in an Western immunoblot for anti-Histag. Cell cultures were grown to OD₅₀₀~0.6 and then incubated either in 16°C for 16 hours or at 37°C for 3 hours. Western immunoblot was conducted from the soluble protein fraction of lysed cells (Figure 1). Stronger Cascade expression with Strong RBS site (S) was visible when cells were incubated in 16°C for 16 hours, then at 37°C for 3 hours. However, Cascade with medium (M) and weak (W) RBS sites were expressed stronger in both conditions than with a strong RBS site. This may have to do with experimental error, or in this particular construct a strong RBS is expressed weaker than the other two. The last two lanes are a negative control of a BL21-DE3 E. coli strain without any plasmids.

Figure 1. Cascade Western immunoblot. M-molecular mass marker (Spectra broad range protein protein ladder); K - possitive control of Csy3 protein; Cascade biobricks were expressed with strong (S), medium (M) or weak (W) RBS sites. BL - BL21-DE3 strain with no transformed plasmids.

E.coli genome targeting with Cascade

We devised an experiment, with which we can see our systems killing profficiency. So we prepared an BL21-DE3 E. coli cells with our Cascade expression construct, along with a complement plasmid, carrying the Cas3 gene in a pCola-Duet vector. Then the whole experiment was to transform the third plasmid harbouring the crRNA region into these bacteria. We transformed two different homogenous crRNA regions, which targeted an essential gene of E. coli genome: DNA pol. III delta subunit (SP1) and RNA pol. alpha subunit (SP2). With these three components present in a bacteria, the killing mechanism turns on when IPTG is present in the medium (pColaDuet vector harbouring Cas3 needs IPTG for expression). The expressed Cascade complex proteins assemble with the synthesized crRNA molecules, and binds to either the DNA pol. (SP1) or RNA pol. (SP2) genes. Then Cas3 is recruited to hydrolyse these genes, ant these bacteria die. After counting of bacteria CFU’s (colony forming units) (Figure 2), we estimated, that under conditions with no IPTG present - all cells showed similar bacterial count with our control (K), which had no crRNA region. This result was expected, considering, that IPTG is needed for Cas3 expression. However, when IPTG is present, we can see a dramatic cell count drop of almost 100fold in bacteria, harbouring our crRNAs (SP1 and SP2), compared to the control (K). From this data we can say, that our Cascade complex biobrick (BBa_K1773022) is expressed and actively functions in degradation of cell DNA.

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Construction of biobrick

Using Biobrick standart assembly method we cloned pLux/cI right promoter and a strong RBS site to the Cascade complex gene cassette. Our aim was to have different expression levels of cascade complexes, we also constructed Cascade complexes with other expression levels: BBa_K1773023 ; BBa_K1773024.

Sources

1. Gasiunas G, Sinkunas T, Siksnys V (2014) Molecular mechanisms of CRISPR-mediated microbial immunity. Cellular and molecular life sciences : CMLS 71: 449-465

2.Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annual review of biochemistry 82: 237-266