Part:BBa_K1773024

Cascade with pLux/cI right promoter and weak RBS

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

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 1887
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2404
  

Usage and Biology

Cascade (CRISPR Associated Complex for Antiviral Defence) complex is an essential component of I-type CRISPR-Cas systems. 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, witch 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 recognisable PAM sequence of this specific I-F Cascade complex from Aggregatibacter actinomycetemcomitans recognises 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 recognises the DNA target, then Cas3 (BBa_K1773003) is recruited for DNA degradation.

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_K1773022.

Expression analysis

Note: This experiment was also done with BBa_K1773023 ; BBa_K1773022 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 16hours, or at 37°C for 3 hours. Western immunoblot was conducted from the soluble protein fraction of lysed cells (Figure1). Stronger Cascade expression with Strong RBS site (S) was visible when cells were incubated in 16°C for 16hours, than 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.

Figure1. 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.