Team:Vilnius-Lithuania/Design

Project design

Regulatory unit cloning

Firstly, we synthesized a pair of oligonucleotide primers, which code for pLux/cI (I1051) promoter sequence. We amplified this promoter sequence with the help of polymerase chain reaction (PCR). These promoters were further cloned into LuxI (K805016) and LuxR (I0462) genes.

We performed one more cloning: pLac operon (R0010) was cloned nearby cI repressor (P0151), which was further cloned into LuxI construct. Finally, we cloned cI+LuxI construct into a plasmid with a LuxR gene. This is our regulatory unit, which, in fact, has all the necessary elements to function and control Coliclock system.

Functional unit cloning

Firstly, we synthesized pLuxI/cI promoter sequences, togerther with different RBS sequences: strong (Bba_B0030), medium (Bba_0032) and weak (Bba_0030). Overlapping oligonucleotides were synthesized to the full constructs during PCR. Functional unit, as mentioned before, consists of three main constructs: Cascade complex, cas3 protein and homogenic CRISPR region. CRISPR regions were synthesized by gene synthesis, while Cascade and cas3 proteins were cloned in our laboratory earlier.

In wild type CRISPR-Cas system proteins we found restriction sites, that are recognized by the restriction enzymes used in Standart Assembly. We aimed to mutate these restriction sites. Cas3 gene had 3 different restriction sites of our interest – EcoRI, XbaI and PstI. Cascade complex had only one restriction site – EcoRI. We performed Quick change gene mutagenesis with the Invitrogen Multi site mutagenesis Plus kit. We changed one nucleotide in that way, that the amino acid, which is coded in the triplet, would not change, however, the restriction site would be damaged. For that reason restrictase would not be able to recognize the site and the middle of the gene would not be cut, when cloning via standart assembly. We sequenced the genes after mutagenesis to make sure that it was successful.

Diagram 1. Cascade after mutagenesis. There is no more EcoRI restriction site, so restriction enzyme should not have any effect on Cascade protein.

After that, our team performed PCR reactions, during which we added prefix and suffix sequences to our genes. We also cloned pLux/cI promoters (along with different RBS sequences) into these genes. All in all, we got CRISPR-Cas system genes, which have different expression pattern. By regulating the expression of functional unit we will control the lifetime of Coliclock.

References

  • Gasiunas G, Sinkunas T, Siksnys V (2014) Molecular mechanisms of CRISPR-mediated microbial immunity. Cellular and molecular life sciences: CMLS 71: 449-465.
  • Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annual review of biochemistry 82: 237-266.
  • Sinkunas T, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V (2011). Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. The EMBO journal 30: 1335-42.
  • Brian J. Caliando, Christopher A. Voigt. Targeted DNA degradation using a CRISPR device stably carried in the host genome. Nature Communications 6, Article number: 6989