Design 1: nsp1,2,3 + nsp4-11 + orf1b + S-3a + rest

This design is the most straightforward for splitting the genome into five segments almost evenly.

After infection, the pseudo-virus will not be able to generate a functional RdRp due to the lack of the main protease and several key proteins for the replication complex.

Design 2: nsp1-4 + nsp5-16 + S-orf3a + rest

After infection, the pseudo-virus will not be able to generate double membrane vesicle or suppress host innate immunity. But viral transcription and structure protein translations are possible if all plasmid products with the 3'UTR can be packaged.

Experimental helper plasmids

Three fluorescent proteins plasmids are designed:

covGFP and covTdTomato can be used to test whether the 5'UTR of S-3a can lead to successful gene expression and whether 5'- and 3'-UTRs of the viral genome can lead to successful viral packaging.

covRestYFP: can be used to detect whether gene N can be expressed, which requires the successful assembly of the viral transcription/replication complex.

Due to funding reasons, it is still not completely clear yet whether the project should be completely open (that needs huge donations) or semi-open with protected intellectual properties (that guarantees the benefit of investors). Patent laws are ancient legacy that work against open scientific research. So, now it is still not possible to describe it clearly in public. What I can say here is that the above vaguely defined 12 plasmids have been ordered. Most of them each expresses a portion of the viral genome. Upon co-transfection into cells (such as Vero cells or HEK293 cells), they will express viral RNAs and proteins, which hopefully will automatically assemble into viral particles in two to three days. You can use the supernatant to infect new cells. If successful, the new cells will show fluorescence (you will need a fluorescent microscope or FACS machine, and this is the most important point to test). Additionally, viral tither should be tested. If it is possible deep sequencing of the RNAs packaged into the viral particles should also be done to know which and how many of each plasmid can be packaged into viral particles. Protocol details can be filled in later by referring to literature and possibly need optimization. You can read a more detailed description here: https://osf.io/grudn/wiki/home/?view_only=ec3c3fca62b54052999dabbeadc8b021 You can register there and ask me to add you into the project.

Nebangwa Derrick Neba Do you have any idea why I cannot add you into this project? Do you need to follow me or something?

Jiang-Nan Yang I am impressed by the detail and novelty of this project. I think its very feasible although equally seem really expensive. I need some time to digest the details before keeping you updated with how I can be able to help based on funds and available equipment.

I followed this project already - I guess it must be the reason you weren't able to add me to the project.

Talk to you soon.

Nebangwa Derrick Neba You may need to follow me personally instead of the project to be added into the project.