TITLE: Filter-paper based PURExpress detection of ZikV and CoV2  RNA by enhancer toehold (TacToe) sensors 
AUTHORS: Tanvi Kale, Chaitanya Anil Athale

[DESCRIPTION]
This method details the approach to using in vitro transcription-translation (TxTr) systems based on PURExpress adsorbed on a paper disc. These to run a toehold detection reaction for COVID RNA. The work is related to a manuscript titled "Translational Enhancer Based Ampli cation of Toehold Sensors (TacToe) for Improved Sensitivity and Speed of Viral RNA Detection" to appear in ACS Synthetic Biology.

[BEFORE_START]
Before you start you need the DNA sequences that are provided in the attached files.

[STEPS]
SECTION: Reaction Kinetics using PURExpress
1. BSA treated paper disk preparation:
1) Make 5% BSA solution in ultrapure water
2) Soak filter paper disc (Whatmann filter paper 42, ashless, dm 42.5) in BSA solution, keep it at room temperature for overnight in a beaker 
3) Rinse the filter paper using ultrapure water (repeat the process for 5 times)
4) Let filter paper dry at room temperature (overnight) or at 65 ºc in hybridizer (~1-1.5h)
5) Use sterile 2mm/3mm biopsy punch to create small paper discs 
6) Store the discs in sterile petri plate
SECTION: Reaction Kinetics using PURExpress
3. Prepare the following reaction mix (Master-mix):

DNA working concentration: 
ZIKV:
Toehold: 24nM
Trigger: 54nM

COV:
Toehold: 0.3nM
Trigger: 8nM

Components	   Working Conc.	    Blank(μl)	     Test(μl)
Buffer A*		                                        2.01	                 2.01
Buffer B*		                                          1.5	                  1.5
CPRG (20mg/ml)	0.6ug/ml	                 0.21	         0.21
Rnase inhibitor		                          0.5                   0.5        
Toehold DNA                     (Adjust depending on the yield and the reaction requirements)
Trigger DNA		           (Adjust depending on the yield and the reaction requirements)
Water		                                         2.78	            -
Total Volume	                                                         7	                        7


*Buffer A and B are the two mixes from NEB's PURExpress

Buffer B		                                          1.5	                  1.5 on ice while preparing the master-mix

 
 
SECTION: Reaction Kinetics using PURExpress
4. From the 7 μl master-mix, pipette out 2μl on each paper-disk placed in a 384 well plate to get triplicates of each reaction (2μl x 3).
 
SECTION: Reaction Kinetics using PURExpress
5. Measure absorbance at 570nm over 100 minutes at a 1 minute interval in a plate reader incubated at 37℃
SECTION: Reaction Kinetics using PURExpress
2. PCR amplification of the sensor and the trigger from purified plasmids

The Sensors and triggers are amplified using the following primers:
 
Following are the lengths of the amplicons (linearised sensor/trigger):
 
 
 
SECTION: Reaction Kinetics using PURExpress
6. Freeze drying disks with CFE (Home-made or PURExpress)+ CPRG

1. Cell free reaction (Mastermix + CFE) applied on paper discs (3mm)(Mastermix 1.02ul +CFE 0.44ul)
2. Air dry
3. Flash freeze in liquid Nitrogen 
4. Put  the disc in PCR tubes (1disc/tube). Make 4 small holes on capusing sterile syringe.
5. Put the tubes in lyophilizer @-75 ºC, 4 hour , 0.04 mbar pressure 
6. Samples store @Room Temperature in sterile falcon with silica inside 
7. Rehydration: Plasmid (~24nM) + water  (0.54ul)