Starting a thread for new #SARSCoV2 ideas that we have been brainstorming amongst friends. Calling for inputs, suggestions and help getting someone to try developing in the lab if it makes sense.
First one now is a yeast/bacteria based diagnostic test
#sciencetwitter
First one now is a yeast/bacteria based diagnostic test
#sciencetwitter
One main constraint in current RTPCR/serology tests is supply of physical materials (RNA extraction reagents etc) and trained personnel. Need POC quick tests. What about a microbe that gives a colorimetric readout? Interested? Read on..
The virus uses both ACE2 (receptor) and TMPRSS2 (protease) to enter human cells. Presumably, and this I dont know for sure, the virus is bound to both simultaneously (atleast for some fraction of time). Critical to the idea 
Now, what if we combine this w a split protein system? ie., make yeast/bacteria express ACE2 and TMPRSS2 (or some truncated, inactive version) on the surface, each tagged with halves of an enzyme?
No virus = inactive enzyme
No virus = inactive enzyme
When virus present in sample, virus brings the two together and reconstitutes active enzyme! If enzyme output can be coupled to a colorimetric output, then that makes for a POC diagnostic.
Ship engineered yeast w enzyme mix substrate in tube, patient takes swab, dips into solution and waits to see colour (or hopefully not). Easy to mass produce, no special things needed. Grow L of microbe and package and ship.
Has issues of course: Live microbe, post test disposal issues, etc. Will need to be dealt with, not an idea killer IMHO.
But lets focus on the science for now.
But lets focus on the science for now.
Structure of ACE2-viral RBD known, so insertion site of one half of enzyme ✔
Structure of TMPRSS2 or structure of TMPRSS2🙁 => need to screen; a split GFP+FACS based screen should help?
Enzyme options? DHFR coupled to NADPH something?
Structure of TMPRSS2 or structure of TMPRSS2🙁 => need to screen; a split GFP+FACS based screen should help?
Enzyme options? DHFR coupled to NADPH something?
Please critique, add thoughts, suggestions. And spread the word to those w lab access
@HarmitMalik @sunillaxman @sunishk @MolBiophysUTSW @JefBoeke @ProfTomEllis @ProfTomEllis @srikosuri @JamesLingford @DeepaAgashe @ms_chirps @reeselab1
@HarmitMalik @sunillaxman @sunishk @MolBiophysUTSW @JefBoeke @ProfTomEllis @ProfTomEllis @srikosuri @JamesLingford @DeepaAgashe @ms_chirps @reeselab1
@FrezzaLab @angie_rasmussen @zeusisdead @TheNunJay @BallouxFrancois @CT_Bergstrom @florian_krammer @VirusesImmunity
Please tag any protein scientists, virologists, yeast people anyone who might be able to help develop this. #colorimetricPOCassay #idea
Please tag any protein scientists, virologists, yeast people anyone who might be able to help develop this. #colorimetricPOCassay #idea
Looping in an older idea that's NGS based, do share if this still makes any sense.. primarily we, a bunch of us, tried to circumvent RNA extraction using microarray kind of idea, and barcoding to pool+sequence.
DHFR doesn't have disulfide bonds, so that's good => it can work in oxidizing environment. Robustness to buffer conditions/additives needs evaluation
And yes, this test will detect other CoV too. Can probably be circumvented by engineering mutations into ACE2 binding interface?
And yes, this test will detect other CoV too. Can probably be circumvented by engineering mutations into ACE2 binding interface?
Collating novel, simple ideas here for covid POC diagnostics - first idea yeast/bacterial colorimetric assay, another w NGS. More ideas to be updated. Please help refine & share w yeast/protein engineering labs that are functioning, thx
Collating novel, simple ideas here for covid POC diagnostics - first idea yeast/bacterial colorimetric assay, another w NGS. More ideas to be updated. Please help refine & share w yeast/protein engineering labs that are functioning, thx
Updating the thought here - rather than cell surface expression, could make 2 independent strains with cytosolic expression of constructs - replacing both ACE2 and TMPRSS2 with nanobodies to RBD (avl in lit), and mix cell lysates when adding sample. Lower b/g, higher sensitivity
This can also be repurposed for serology testing - replace nanobody 1 with viral antigen, and nanobody 2 with anti-IgG/anti-IgM secondary antibody, with the antibody in the patient sample binding both and thus enabling complementation of enzyme halves
Theoretical sensitivity is 10^4 virions, possibly 10^3 with luck. As good as the RT PCR, but doable at home and producable at scale. Every human on earth can test every day for 2 weeks and we can still make more quite quickly.
