DNA sequence for detecting normal and phosphorylated tau(p-tau)

to find my work on kernel you can search the Mustafa Alwash htbaa 2026 repo which is at Adrian Filips account provided by openbioscience node.

Defining the Expression Target

i have built two split protein sensors for both normally structured tau protein and phospholyted tau(p-tau) protein, here is a structured template of what is happening in my repo:

Total Tau beta-lactamase sensor

this is the 1st sensor made for detecting normal tau in the blood. Its a split protein made up of two parts , each part follows the same general scheme which is {backbone}→{promotor}→{RBS}→{a nanobody}→{linker}→{subunit}→(terminator):

split protein A

you can see this part in this construct ,named tau, which responsible for binding to the C-terminal of tau.

split protein B

this part is at this contruct ,named tau B.

p-tau sensor

this is the 2nd sensor made for detecting phospholyted tau proteins in the blood. To be specific, this will detect only the molecules that are phosphorylated at serine 422 , whch is a type of p-tau that’s linked to Al-zheimer’s disease

split A

this constract ,named p-tau A, will be the part responsible of attachment with the phospho part of the tau

split B

is the same as the Total tau B split this constract ,tau B.

Host / Chassis: E. coli SHuffle T7 strain (for engineered redox cytoplasmic folding) and E. coli cell-free protein synthesis (CFPS) lysate.

Localization: Cytoplasm / Cell-Free Reaction Solution. No signal peptide or secretion tag is required as the reaction occurs in cell-free lysate.

desgning the genetic parts

Assembling the constructs

All split protein constructs were assembled in Kernel using the same $5’ \rightarrow 3’$ orientation scheme:

{backbone} -> {promotor} -> {RBS} -> {nanobody} -> {linker} -> {reporter} -> {terminator}

Assembled constructs in Kernel

1. Normal Tau Sensor - Split Protein A (LargeBiT):

   • {pSB1C3} -> {T7} -> {RBS A1} -> {H3-2} -> {(G4S)3} -> {BBa_I757012} -> {T7 Terminator}
   • construct

2. Normal Tau Sensor - Split Protein B (SmallBiT):

   • {pSB1C3} -> {T7} -> {RBS A1} -> {MT3.1} -> {(G4S)3} -> {BBa_I757011} -> {T7 Terminator}
   • contruct

3. pTau S422 Sensor - Split Protein A (LargeBiT):

   • {pSB1C3} -> {T7} -> {RBS A1} -> {pS422} -> {(G4S)3} -> {BBa_I757012} -> {T7 Terminator}
   • constract

4. pTau S422 Sensor - Split Protein B (SmallBiT):

   • {pSB1C3} -> {T7} -> {RBS A1} -> {MT3.1} -> {(G4S)3} -> {BBa_I757011} -> {T7 Terminator}
   • contruct

Testing plan and experimental assay

To test and validate the biosensor constructs, the system will be run in an E. coli Cell-Free Protein Synthesis (CFPS) assay

1. Cell-free expression setup Combine E. coli CFPS extract, master mix (NTPs, amino acids, energy regeneration mix, T7 RNA polymerase), and plasmid DNA (5-10 ng/µL). Co-express the sensor pairs:

Misfolded Tau pair: {H3-2-LargeBiT} + {MT3.1-SmallBiT}

pTau-S422 pair: {pS422-LargeBiT} + {pS422-SmallBiT}

Incubate at 30°C for 2-4 hours for transcription and translation.

2. Target titration and signal measurement

Add Nano-Glo substrate (Furimazine) at a 1:50 dilution to the CFPS mix.

Spike in purified target protein across a concentration gradient from 0 nM to 1 µM:

Aggregated Tau vs. monomeric Tau for the misfolded Tau sensor.

Synthetically phosphorylated Tau (pS422) vs. unphosphorylated Tau for the pTau sensor.

Record luminescent intensity at 460 nm on a plate reader over 30 minutes.

3. Experimental controls Background control: Express single constructs alone (LargeBiT only or SmallBiT only) without their complementary half to measure baseline background assembly.

Specificity control: Incubate the biosensor pairs with non-target proteins (BSA or unphosphorylated monomeric Tau) to confirm no off-target activation.

Positive control: Express full-length, intact NanoLuc driven by the {T7} promoter to set the maximum luminescence baseline in lysate.

Success metric: Target a Signal-to-Noise ratio (S/N = Luminescence_target / Luminescence_blank) greater than 10.

Project unknowns and potential risks

1. Linker spatial constraints

• The (G4S)3 linker length might not match the physical distance between Tau epitopes.
• If too short, LargeBiT and SmallBiT cannot reach each other; if too long, background noise increases.

2. Nanobody folding in cell-free lysate

• VHH nanobodies require disulfide bonds to fold properly.
• Standard E. coli lysate is reducing, which could lead to misfolded or inactive nanobodies.

3. Stoichiometry and background assembly

• Controlling equal co-expression of both plasmids in lysate is difficult.
• Excess uncomplexed reporter halves can cause non-specific background signal without target Tau present.

AI prompts

• I did relay on gemini when searching for eligible candidates for nanobodies for the detection of both normal and phosphorlyated tau protein , if not the search can go on for weeks
• Also, It did help with the concentrations and amounts mentioned in the “Testing plan and experimental assay” part of the HW