From a meeting with Dr. Peter Nguyen from the Wyss Institute, an expert in applying freeze-dried cell-free systems in advanced materials, he clarified my questions about the lab design and kindly provided detailed feedback on my Plasmid Design, specifically Zinc-LacZ sensor.
Moving foward, I will focus my research on developing a zinc-LacZ sensor through step-by-step testing, while maintaining zinc-sfGFP sensor and pH sensor as parallel alternatives.
Below is a diagram of the complete zinc-LacZ sensor circuit design:
As Peter suggest, I will test this design by breaking it down into parts:
the whole list of sensors is as follows:
| sensor | reporter | part? | test purpose | plasmid | cell-free reaction | benchling sequence |
|---|---|---|---|---|---|---|
| zinc sensor | LacZ | reporter only | test if LacZ is always on without ZntR (leakage) | Puc19 (Backbone) - |
[PzntA (promoter) - B0034 (RBS) - LacZ (reporter) - B0015 (terminator)] | plasmid + TXTL + CPRG + E. coli RNA polymerase | link | | zinc sensor | LacZ | whole sensor | test if PzntA is always on without Zinc | Puc19 (Backbone) -
[T7 (promoter) - B0034(RBS) - ZntR (activator) - B0015 (terminator)] -
[PzntA (promoter) - B0034 (RBS) - LacZ (reporter) - B0015 (terminator)] | plasmid + TXTL + CPRG + E. coli RNA polymerase | link
| | zinc sensor | LacZ | whole sensor | test if Zinc can turn sensor on | Puc19 (Backbone) -
[T7 (promoter) - B0034(RBS) - ZntR (activator) - B0015 (terminator)] -
[PzntA (promoter) - B0034 (RBS) - LacZ (reporter) - B0015 (terminator)] | plasmid + TXTL + CPRG + E. coli RNA polymerase + zinc concentrate | same plasmid as above, just different condition (without / with zinc) | | zinc sensor | sfGFP | whole sensor | test if replacing LacZ with sfGFP (a much shorter reporter then LacZ) works | Puc19 (Backbone) -
[T7 (promoter) - B0034(RBS) - ZntR (activator) - B0015 (terminator)] -
[PzntA (promoter) - B0034 (RBS) - sfGFP (reporter) - B0015 (terminator)] | plasmid + TXTL + E. coli RNA polymerase + zinc concentrate | link | | pH sensor | sfGFP | whole sensor | jut in case zinc sensor dont work | Puc19 (Backbone) -
[T7 (promoter) - B0034 (RBS) - sfGFP (reporter) - B0015 (terminator)] | plasmid + TXTL + trehalose + RNase inhibitor + different pH buffers (?) | link |