We build biobricks for our "Multivirus Monitor" project. These parts cover all aspects in our project, including sample processing, Cas13 protein expression and purification and fluorescence detecting.
Cas13 protein homologues
To achieve our multivirus detecting goal, we choose LwaCas13a, LbaCas13a, PsmCas13b, CcaCas13b proteins as our detecting effectors which are found in Leptotrichia wadei, Lachnospiraceae bacterium NK4A179, Prevotella sp.MA2016, and Capnocytophaga canimorsus respectively. To obtain these proteins with high purity and activity, we designed biobricks to express the four Cas13 proteins homologues. Meanwhile, we attached 6×His/Twin strep tag to make it can be purified by Ni-NTA purification. Besides, we added SUMO/MBP tag to increase the solubility of the proteins. These biobricks are under control of T7-promoter and Tphi-Terminator. Finally, we created our favorite part:
Involved basic parts in this construct are
We characterized these parts and documented them in the part registry. For more information on the design and characrization of the parts, please click the part number listed below.
Primers for Recombinase Polymerase Amplification (RPA)
To amplify the virus signal, we used RPA technology to amplify the viral RNA. Different from primers of PCR, the RPA primer lengths should be ~25–35 nt, and the total amplicon size should be 80–140 bp. Primers are typically designed with melting temperatures between 54 °C and 67 °C. In addition, because we need to do transcription experiments downstream, a T7 promoter was appended to the 5ʹend of the forward primers to trigger the transcription.
Viral characteristic sequence
To achieve the purpose of specific detection, we built BBa_K3406010, BBa_K3406011, BBa_K3406012 , BBa_K3406013 which contains viral characteristic sequence. Besides, we construct BBa_K3406014 which enables the transcription of viral RNA to simulate the real virus.
crRNA for Cas13 protein homologues
The crRNA is an oligonucleotide chain, it can combine with Cas13 protein and guide Cas13 to find the target viral sequences. It consists of two parts, DR sequence and spacer. The DR sequence is determined by Cas13 protein, and different Cas13 proteins have different DR sequences. And the spacer is determined by a specific target. We built BBa_K3406015 , BBa_K3406016 , BBa_K3406017 and BBa_K3406018 to guide our four Cas13 protein target SARS-CoV, MERS-CoV, SARS-CoV-2 and H1N1.
Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018 Apr 27;360(6387):439-444. doi: 10.1126/science.aaq0179. Epub 2018 Feb 15. PMID: 29449508; PMCID: PMC5961727.
BBa_K2323004 Lwa Cas13a under T7 promoter with solubility tag.Designed by: Aurore Dupin Group: iGEM17_Munich