Experiments

Cell-Based Experiments

MFalpha1

The yeast strains used for transforming our parts have a GFP expressing gene that is induced through the yeast mating systems. This means that transcription is activated by either adding the mating factor alpha peptide, which activates the yeast mating system, or by using a LexA-fused transcription factor.. When MF(alpha)1 is expressed, the mating factor alpha peptide will activate the mating system, inducing GFP expression. This GFP expression can be quantified by measuring the fluorescence. This will be done using a plate reader, compared to a sterile control and the yeast strain not containing our MF(alpha)1 assembly.

Zinc-finger DNA Detection

Zinc-finger-complex strains will be tested by adding the target DNA to the cell culture and measuring the fluorescence. The Zinc-finger strain will release the transcription factor that will bind to the GFP promoter, thereby activating its expression. Like in the MF(alpha)1 test, the expression of GFP will be measured using a plate reader. This will be done by doing twofold serial dilutions of the target DNA so that a smallest detectable target DNA concentration can be identified.

Violacein Synthesis

Violacein expression will be tested by growing strains transformed with all the genes required for the biosynthesis pathway (vioABCDE). If the system works, the cell cultures should be visibly purple.

To test if violacein can be induced through the mating system, a strain with one of the genes in the biosynthesis pathway will have a promoter that is induced by the mating system (LexO). By adding the mating peptide (mating factor alpha peptide) to the culture, it should subsequently induce the expression of the violacein gene. This will thus activate the synthesis pathway leading to the synthesis of violacein, producing a visible purple readout.

The Complete Cell-Based System

The full system will be tested by adding the target DNA to a yeast strain culture with the complete system. This will be done similarly to when testing the zinc-finger DNA detection. However, the complete system should produce the purple violacein pigment insead of GFP. This can be detected using absorbance measurements in a plate reader or visually by eye.

Protocols

• DNA primer protocol
• E. Coli transformation
• Gel electrophoresis
• E. Coli innoculation
• LB plate preperation
• Primer Mixutre preperation
• Yeast Colony PCR
• Yeast MoClo Workflow
• Primestar PCR
• Phusion PCR
• Yeast Transformation
• GeneJET Plasmid Miniprep
• GeneJET PCR Purification
• GeneJET Gel Extraction
• DNA primer protocol
• DNA primer protocol

Cell-Free Experiments

FRET Protein

With the use of a plate reader it can be established that if FP (FRET Protein) is expressed in S. cerevisiae in by comparing it with the same untransformed strain. With this it can be known if FP is correctly integrated and transcribed, and it will likely be able to be cut by Tobacco Etch Virus Protease (TEVp) as it has a recognition site for TEVp in the linker that fuses the fluorescent proteins together.

TEVp

If FP is validated to be expressed, then the addition of TEVp would change the fluorescent properties of FP. The fluorescent proteins would be no longer forced to be in eachothers proximity and as such FRET would no longer occur to the same extent. Meaning, if the fluorescent properties of FP changes with the addition of TEVp, then it is reasonable that TEVp is expressed and functional.

This can be done inside a cell that contains both parts, using a cell with FP and a cell with TEVp as negative controls. Or it can be done by purifying these proteins and then mixing them just before the experiment. This would be a process that would not be instant. As such it should be measured over time in a plate reader with FP and TEVp in separate wells as two different negative controls.

iGal

If TEVp works, then it should cut Inhibited Beta-galactosidase (iGal) if that protein is folded as intended. By mixing X-gal + TEVp + iGal, the test should turn blue if iGal is functional. Three negative controls should be made, the first one with just TEVp, the second one with just iGal and the third one with just X-gal. If the third negative control turns blue, then the environment or buffer is the cause. If the second negative control turns blue alongside the test, but not the first and second negative controls, then iGal is not inhibited but rather always active like wild type Beta-galactosidase. If the first negative control turns blue, then there are unknown factors that can degrade X-gal that are beyond what is mentioned here.

Split TEVp

If TEVp works, then it should be validated that it is possible to split the protein into two parts that can later complement each other to regain the catalytic function of TEVp. Using the parts ABI-cTEV and PYL-nTEV, it is possible to induce complementation of ABI and PYL with the addition of Abscisic acid (ABA), which would bring cTEV and nTEV close and induce their complementation [1]. The test would be performed by mixing ABI-cTEV + PYL-nTEV + FP, with and without ABA and using TEVp + FP as a positive control and FP as a negative control. In case of any complication with FP, an alternative would be to replace FP with X-gal & iGal, if iGal is shown to work as intended. The mixing could either be done in vivo or in vitro as described in Validating TEVp. The test would give qualitative information on if the split TEVp can complement each other, and an upper and lower bound on the spontaneous complementation of free floating halves of split TEV. It is important to know if spontaneous complementation of split TEV is a significant phenomena, as in the final test that could result in false positives.

dCas9-Split TEV

If split TEVp works, then it’s a good indication that all dCas9 has to do is to bring the split TEVp together and the system should work. It just needs to be confirmed that dCas9 binds correctly and the whole dCas9-split TEV proteins are folded correctly. By combining FP + dCas9-cTEV and dCas9-nTEV with two guide RNA (gRNA), that directs the binding of each dCas9 close to each other on the same DNA strain + adding target DNA + FP, there should be a measurable change in the fluorescent properties if all is functional. It may be easier to do the test in vivo as gRNA is easily degraded. Using appropriate controls such as the system without target DNA (negative control) and the system with TEVp (positive control) it would be established if dCas9-split TEV is functional. As for Validating split TEVp, in case of any complication with FP, an alternative would be to replace FP with X-gal & iGal, if iGal is shown to work as intended.

Optimizing dCas9-split TEV

If dCas9-Split TEV works, then it would be of interest to find the best combination of gRNA and linker length. Using factorial design of experiments, the number of experiments required to find the best combination of gRNA and linker length would be minimised. The factorial design would have the variables: Distance between the binding sites of the gRNA + linker length + eventual effects from binding-strength of gRNA, off-target binding and spontaneous complementation of split TEV if the first two variables do not explain the results. Or if one has the time, all combinations could be tested as the procedure for one experiment is essentially identical to all others. With this data, it would also be of great interest if it would validate or reject our model.

dCas9-Split TEV - Specificity, Sensitivity and Robustness

For the combinations of gRNA and linker lengths for dCas9-split TEV that give the highest activity, it would be tested how specific the system is by adding control DNA. A mass of “random” DNA, from a soil sample or a lake sample, could be used as control DNA. If the system is not very specific and is activated by many other sequences of DNA, the control DNA could activate the system. If this test would be used in a clinical setting, further testing would be required for a greater variety of control samples, but that is beyond the scope of this project.

The sensitivity would be tested by simply diluting the target DNA and adding it to the test and measuring the time it takes until it gives a significant response. It would be of great interest to test the same concentrations of the target DNA that is naturally found in patients with schistosomiasis at different stages of infection.

The robustness of the test would be if it still works despite there being other components that may disturb it. To test the system with target DNA + control DNA would give information if other free floating DNA may disturb the system. The final test would be to add target DNA + control DNA + saliva - this is to examine if a complex solution such as saliva would disturb the system or not. If that works, the system would have a strong proof-of-concept.

Making it a Cell-Free system

The next step would be to find a reliable way to make the components all cell free. This could be done through protein purification, secretion or lysing the cells with chemicals and adding protease inhibitors (that does not affect TEVp) to protect the proteins in the system. If necessary, the production of gRNA could be done with cell-free transcription as it may be destroyed in protein purification and when lysing the cells, or it may be stuck inside the cell if dCas9-split TEV is secreted. The now cell-free components would only have to be combined with a sample and an appropriate control just before the test is performed.

Sources

[1] Fink, T., Lonzarić, J., Praznik, A. et al. Design of fast proteolysis-based signaling and logic circuits in mammalian cells. Nat Chem Biol 15, 115–122 (2019). https://doi.org/10.1038/s41589-018-0181-6