iGEM 2022 — Team chalmers-gothenburg

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{% block lead %}Check out what each system explored in the wetlab in our notebooks!{% endblock %}
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We present the Cell-based and cell-free notebooks here! They contain a short summary of all lab work that was done each day, with weekly summaries. The notebooks track the progress of the Wet Lab team throughout the project. The relevant and referred to protocols in the Notebooks are found in [Experiments](https://2022.igem.wiki/chalmers-gothenburg/experiments).

[![](https://static.igem.wiki/teams/4367/wiki/notebook/cell-based-system-notebook.png)](https://static.igem.wiki/teams/4367/wiki/notebook/notebook-cell-based.pdf)
[![](https://static.igem.wiki/teams/4367/wiki/notebook/cell-free-system-notebook.png)](https://static.igem.wiki/teams/4367/wiki/notebook/notebook-cell-free-2.pdf)

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Our path through the engineering cycle{% endblock %}
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### Cell-based system

The  **cell-based design**  was developed by first visualising what types of functions a yeast cell would need to give a visual readout from a DNA sample. From the initial brainstorming session we realised that a yeast cell would need the following: A DNA detecting function, an inducible transcription factor, a gene which can produce colour without any substrate and an exponential system to make the readout more clear. To tackle these demands we found zinc-fingers as our DNA detecting molecule, these were coupled to a transmembrane protein. Two of these zinc-fingers were used together with an established membrane yeast two hybrid system using split Ubiquitin attached to the transcription factor LexA-VP16. This would make it so that if a sample of the correct DNA was found by a yeast cell, it would induce the transcription factor LexA-VP16 which would turn on two genes: MF(alpha)1 (a mating factor in yeast) and a Violacein gene (leading to the production of violacein, a coloured pigment which only requires aminoacids to be produced). The mating factor would make it so other yeast cells would also start expressing Violacein and the Violacein would turn the cells visibly violet.

The actual build of the cell-based system started of brilliantly, as the golden gate assembled parts starting forming, spirits were high and the preliminary results showed we had no mutations or misgivings in our construct. However, due to time limitations all the transformations weren't finished in time. The strain for testing the MF(alpha)1 system was finished in time though. The testing was done by measuring GFP expression using a plate reader and the results showed that the fluorescence was higher in these cells, indicating that it worked.

¨
![](https://static.igem.wiki/teams/4367/wiki/bilder/t-linkoping-sweden-engcycleeng.png)

### Cell-free system

The **cell-free system** was designed around a split TEV technique and splitting of the LacZ gene into an alpha fragment and an omega fragment. For the Alpha fragment, we used a previous iGEM teams Part: BBa\_I732006 and the Omega fragment was designed *in silico* from Beta-galactosidase, deleting the specified amino acids according to their instructions [1]. We then fused the fragments with a cleavable linker in opposite order, which produces our Inhibited Beta-galactosidase protein (iGal). The split TEV technique we used was selected once we saw the results of the split luciferase in combination with a paired dcas9 that iGEM Peking 2015 used for tuberculosis detection. The Split TEV technique was taken from an article published in nature and there they had each half attached to two other proteins that could be induced to adhere to each other. [2] Combining these three resulted in our design: a system with a paired dCas9 which would create a functional TEVp to cut iGal to form a functional tetramer which would colour the sample blue in case our target DNA was present. See figure 2.

![](https://static.igem.wiki/teams/4367/wiki/bilder/illustrationer/alpha-schematisk-bild.png)

Building the project with golden gate assemblies was an arduous journey with many pitfalls among them are, PCR protocols that didn’t show results, gel extractions which went wrong, sequencing samples that got lost, and plate reading which were less than ideal. Eventually a final construct was made through the golden gate assembly, first all of the level one plasmids were transformed and grown in *E. coli* after which the level two assembly proceeded and multiplied in *E. coli* again. The parts were then genome integrated using Cas9 and the final construct was assembled in yeast.

Testing the parts of the cell-free system gave strange results, we can see that most samples are blue but not all and the different iterations of iGal and TEV seem to interact in ways that are unpredictable. The next step forward is to perform the experiment with purified proteins as there were unknown factors that made the negative controls give a positive response, which made it not possible to evaluate if the cell-free system works.

## References

[1] http://parts.igem.org/Part:BBa\_I732006
[2] https://www.nature.com/articles/s41589-018-0181-6

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{% block title %}Experiments{% endblock %}
{% block lead %}All the research, experiments, and protocols we've used can be found here{% endblock %}
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[![](https://static.igem.wiki/teams/4367/wiki/notebook/cell-based-system-notebook.png)](https://static.igem.wiki/teams/4367/wiki/notebook/notebook-cell-based.pdf)
[![](https://static.igem.wiki/teams/4367/wiki/notebook/cell-free-system-notebook.png)](https://static.igem.wiki/teams/4367/wiki/notebook/notebook-cell-free-2.pdf)

## 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](https://static.igem.wiki/teams/4367/wiki/protocols/dna-primer-preparation.pdf)
* [E. Coli transformation](https://static.igem.wiki/teams/4367/wiki/protocols/e-coli-transformation.pdf)
* [Gel electrophoresis](https://static.igem.wiki/teams/4367/wiki/protocols/gel-electrophoresis-preperation.pdf)
* [E. Coli innoculation](https://static.igem.wiki/teams/4367/wiki/protocols/inoculation.pdf)
* [LB plate preperation](https://static.igem.wiki/teams/4367/wiki/protocols/protocol-sysbio-plates-and-media-2018-lb-and-ypd.pdf)
* [Primer Mixutre preperation](https://static.igem.wiki/teams/4367/wiki/protocols/primer-mixture-preparation-protocol.pdf)
* [Yeast Colony PCR](https://static.igem.wiki/teams/4367/wiki/protocols/yeast-colony-pcr.pdf)
* [Yeast MoClo Workflow](https://static.igem.wiki/teams/4367/wiki/protocols/protocol-for-yeast-moclo-workflow.pdf)
* [Primestar PCR](https://static.igem.wiki/teams/4367/wiki/protocols/primestar-pcr-protocol.pdf)
* [Phusion PCR](https://static.igem.wiki/teams/4367/wiki/protocols/phusion-pcr-protocol.pdf)
* [Yeast Transformation](https://static.igem.wiki/teams/4367/wiki/protocols/liac-ss-dna-peg-yeast-transformation-version-2.pdf)
* [GeneJET Plasmid Miniprep](https://static.igem.wiki/teams/4367/wiki/protocols/genejet-plasmid-miniprep.pdf)
* [GeneJET PCR Purification](https://static.igem.wiki/teams/4367/wiki/protocols/genejet-pcr-purification-kit.pdf)
* [GeneJET Gel Extraction](https://static.igem.wiki/teams/4367/wiki/protocols/genejet-gel-extraction.pdf)
* 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

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{% block title %}Results{% endblock %}
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## Cell-based Results

The only sub system of the cell based method that was tested was the MF(alpha)1 activation of the yeast mating system. The reason no other subsystems, let alone the full cell based system, was tested was due to issues with cloning, both for *E.coli* and *S. cerevisiae*. Our systems are composed of many parts, both coding genes and different promotors and terminators. Throughout the summer, a lot of assemblies and transformations failed at all different stages. Besides problems with cloning, we were not able to go into the lab until July because of maintenance reducing the time we had available in the lab. By the time we had planned to finish, at the end of August, we had only barely finished making our assemblies. Most of September we spent attempting to finish transforming our assemblies into yeast, while also trying to remake unsuccessful assemblies in E.coli. There was very little time to finish the systems before the wikifreeze.

The MF(alpha)1 subsystem was tested by measuring GFP fluorescence in the yeast strain.
The yeast strain we used had a GFP gene that is activated when the mating system is activated by the mating factor α-peptide.
The MF(alpha)1 gene produces mating factor α-peptide when expressed.
Therefore, the yeast strain with a promotor constantly expressing MF(alpha)1 should also express GFP,
as the mating system is activated by the mating peptide.
Using a plate reader to measure fluorescence showed that the MF(alpha)1 yeast strain indeed expressed GFP,
see table below.

* Issues with cloning, both in *E.coli* and *S. cerevisiae*, not working properly. This was very time consuming.
* Our cloned MF(alpha)1 gene works for activating the yeast mating system in  *S. cerevisiae* , inducing GFP expression.
* Not enough time to clone violacein pathway into yeast, and therefore no experimental data on the violacein system produced.
* NubG and Cub “activation by binding target DNA ….

| Replicate | 1 | 2 | 3 |
| --- | --- | --- | --- |
| MF(alpha)1 | 2938 | 2825 | 2807 |
| D4 | 1757 | 1462 | 1468 |

## Cell-free Results

* The experiment to test if the transformed *S. cerevisiae* expressed FRET failed. The plate reader displayed no difference in fluorescence between the blank, control and the test.

The test to quantify FRET of pair fluorescent proteins (mNeongreen-mRuby3) failed. The blank, control and the *S. cerevisiae* transformed with FRET showed essentially the same fluorescence. This indicates that there was no evidence that FRET was expressed. The colony PCR indicated that the gene had the right length, but the sequencing results were terrible. In the future, it would be good to use common fluorescent proteins, such as GFP, that the available instruments would be able to measure more easily.

* The experiment to test if the designed Inhibited Beta-galactosidase worked gave strange and unsatisfactory results. A conclusion is that something within the lysed cell slurry of *S. cerevisiae* can degrade X-gal.

The experimental setup tested the mixtures of X-gal and cell-lysates from *S. cerevisiae* transformed with corresponding protein is shown in table 1 and table 2. Each column corresponds to one transformation of *S. cerevisiae* with TEV and each row makes up one transformation with the Inhibited Beta-galactosidase.

#### Experiment

Table 1. Experimental setup of in the figures below.

| Date: 2/9 | TEV5.4.2 | TEV5.4.5 | TEV5.4.6 | NO TEV |
| --- | --- | --- | --- | --- |
| iGal\_1.2 | X | X | X | X - TEV |
| iGal\_3.1 | X | X | X | X - TEV |
| iGal\_3.3 | X | X | X | X - TEV |
| iGal\_3.4 | X | X | X | X - TEV |
| iGal\_4.1 | X | X | X | X - TEV |
| iGal\_4.2 | X | X | X | X - TEV || iGal\_4.3 | X | X | X | X - TEV || No iGal | X - iGal | X - iGal | X - iGal | Only X-gal and MQ |

X :10 μL X-gal stock (20 mg/ml), 30 μL iGal lysed cell slurry
, 30 μL TEV lysed cell slurry
, 20 μL MQ

![](https://static.igem.wiki/teams/4367/wiki/bilder/alpharesults/milliq-2-9-at-3-9.jpg)
![](https://static.igem.wiki/teams/4367/wiki/bilder/alpharesults/milliq-2-9-at-4-9.jpg)
![](https://static.igem.wiki/teams/4367/wiki/bilder/alpharesults/milliq-2-9-at-5-9.jpg)
![](https://static.igem.wiki/teams/4367/wiki/bilder/alpharesults/milliq-2-9-at-8-9.jpg)

*Figure 1.Wells containing lysed cells that had been transformed with TEVp and iGal. The wells also contained X-gal and MilliQ as buffer. The pictures are how the experiment looks after 1, 2, 3, and 6 days since its start. The experimental setup is described in table 1.*

Table 2. Experimental setup of mixing TEVp, B-gal and X-gal in PBS as a buffer.

| Date: 3/9 | TEV5.4.2 | TEV5.4.5 | TEV5.4.6 | NO TEV |
| --- | --- | --- | --- | --- |
| iGal\_1.2 | Y | Y | Y | Y - TEV |
| iGal\_3.1 | Y | Y | Y | Y - TEV |
| iGal\_3.3 | Y | Y | Y | Y - TEV |
| iGal\_3.4 | Y | Y | Y | Y - TEV |
| iGal\_4.1 | Y | Y | Y | Y - TEV |
| iGal\_4.2 | Y | Y | Y | Y - TEV || iGal\_4.3 | Y | Y | Y | Y - TEV || No iGal | Y - iGal | Y - iGal | Y - iGal | Only X-gal and PBS |

Y: 5 μL X-gal stock (20 mg/ml), 30 μL iGal lysed cell slurry, 10 μL TEV lysed cell slurry, 100 μL PBS

![](https://static.igem.wiki/teams/4367/wiki/bilder/alpharesults/pbs-3-9-at-4-9.jpg)
![](https://static.igem.wiki/teams/4367/wiki/bilder/alpharesults/pbs-3-9-at-5-9.jpg)
![](https://static.igem.wiki/teams/4367/wiki/bilder/alpharesults/pbs-3-9-at-8-9.jpg)

*Figure 2. Wells containing lysed cells that had been transformed with TEVp and iGal. The wells also contained X-gal and PBS as buffer. The pictures are how the experiment looks after 1, 2, and 5 days since its start. The experimental setup is described in table 2.*

#### Experiment description

Our designed Inhibited Beta-galactosidase (iGal) should become active when it comes into contact with TEV protease (TEVp). Only an activated iGal would turn X-gal into a blue pigment, indicating a positive response. The experiment was designed to express these proteins inside different *S. cerevisiae* cells, and then to lyse the cells to get the extracted proteins into contact. The cells were lysed and added into wells with X-gal with MilliQ water as buffer. Three types of negative control were also included. The first negative control was TEVp + X-gal which checked that the lysed cells didn’t naturally break down X-gal. The second negative control was iGal + X-gal, which checked that iGal didn’t break down X-gal before it got into contact with TEVp. The third negative control being only X-gal was to check that X-gal didn’t naturally break down in the buffer or other environmental conditions. It was observed that some of the wells containing the lysed cell mixtures turned blue, but so did the first and second negative controls. The experiment was run again in a similar fashion again, but changing the buffer from MilliQ to PBS. The result was similar, with some of the tests turning blue and the first and second negative controls also turning blue.

#### Discussion

As the first negative control turned blue, but not the third, and since TEVp does not degrade X-gal, this result indicates that there is something within lysed cell slurry of *S. cerevisiae* that can degrade X-gal, without any iGal or Beta-galactosidase needing to be present.

There’s a risk that iGal would not function as intended, that it would always be active rather than only becoming active when it comes into contact with TEVp. This would cause every test to become blue as well as the second negative control. Although, with Occam's razor as a principle, there already is the explanation above for why the first negative control turns blue. If the first negative control is blue, then it is because the lysed cells are breaking down X-gal, which would mean that the second negative control would also turn blue. This is because the cells used in the tests came from the same *S. cerevisiae* cultivation. This means that there is no evidence to confirm that iGal is not functional as both controls were blue and not only the second control.

No obvious pattern alongside any column or row (eg. any particular version of TEVp or iGal) could be observed. The effect of mixing the cell lysates did not consistently make the test more blue than the controls, indicating there is no obvious effect from mixing these. If it is assumed that the second-order effect of adding TEVp-lysate and iGal-lysate is zero, the effect of each addition should give a linear response. Although, no obvious linear effect could be observed. If the effect was linear, the tests would simply be a combination of the “blue-ness” of the controls, but in some cases, the controls are more blue than the tests. This indicates that there is some other unaccounted factor that caused the X-gal to degrade and the assumption that only TEVp, iGal, X-gal and a cell-lysate was added doesn’t explain the results. It is as if different things are pipetted up from the same tube, despite it only containing one type of cell-lysate, TEVp or iGal.

One possible explanation for the results is that the content within the tubes containing the lysed cells had sedimented into different layers, with some layers that contained something that could degrade X-gal. If this was the case, the depth that one would pipette at would determine if the compound that could degrade X-gal would get transferred to the well or not. Meaning if one would pipette from the bottom of the tube, this might cause the well to have turned blue. This is not too unreasonable of an explanation, as the cell lysate was never centrifuged and the supernatant wasn’t separated from what would be the pellet.

In either way, there was no obvious effect from mixing the cell lysates, indicating that the tests did not work as desired. This could be because the genes had failed to be expressed, had mutated and were non-functional, had been degraded by other proteases, the buffer not allowing it to function properly, misfolding of iGal such that the cut-site for TEVp wasn’t exposed or misfolding inducing loss of function. Or simply being that even though TEVp had cut iGal it is unable to form the tetramer, or that iGal is enzymatically non-functional in the first place. As the tests didn’t show any effect from mixing the cell lysates, the combination of errors and time constraints didn’t allow us to troubleshoot and test the system further.

## Replication of the experiment

If one were to replicate this experiment, one would ideally use protein purification to separate TEVp and iGal from the cell lysate. If that option isn’t available, it would be good to use protease inhibitors that don't affect TEVp, to keep the TEVp and iGal from being degraded. It would be recommended to centrifuge the cell lysate to potentially exclude the compound in *Saccaremyseas cerevisiae* that degrades the X-gal. TEVp requires a reducing buffer to be able to function, so it may be necessary to add that to the test. It would also be good to use larger volumes of liquid as the water evaporates and changes the volume significantly. Some wells almost dried out for the test that started on 2/9, as the volume was 90 μL. X-gal is light sensitive, so storing it in a dark room or encased in aluminium foil is advised. Heat could degrade X-gal, but in the timespan of a few days in 30 degrees and in MilliQ water or PBS, no noticeable degradation of X-gal could be seen by eye in this test as it didn’t turn blue. This test used X-gal, although it was simply suspended in MilliQ and not fully dissolved. It would be better if the X-gal would be dissolved in something and this solvent or buffer should be selected such that it does not denature the proteins.

With the results from AlphaFold on the structure of iGal, there exists a risk that it is always active. The reason for this is that a linker is possibly too long and could allow for the Alpha and Omega fragments to complement each other despite the linker not being cleaved. If this would be the case, iGal would have to be redesigned, possibly by just shortening the linker. So if the test would be performed in the future and it would display positive results with just iGal and X-gal, this could be a solution. It would be a good idea to use AlphaFold to see what length of the linker would not result in the Alpha-fragment reaching the spot on the Omega-fragment that would result in alpha-complementation.

If the test would display negative results it would be good to test if TEVp is functional. With fluorescent proteins and the right tools, it could be easy to see if TEVp functions or not by measuring if the FRET phenomena is happening. It would be relatively easy to know if the fluorescent proteins are expressed and possible to quantify it with a plate reader. If the addition of TEVp would decrease the FRET-phenomena more than a control, it’s a clear sign that the TEVp functions. If TEVp functions, but the test is negative, it could imply that the problem lies with iGal and not TEVp. All this means that using fluorescent proteins is a good way of error-searching as it is relatively easy to know if they function or not with the right tools.

## Future experiments

View the page  ["experiments"](https://2022.igem.wiki/chalmers-gothenburg/experiments) to find out what future experiments would look like.

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