iGEM 2023 — Team uioslo-norway

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

## Notebook

---

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block page\_content %}

## Engineering Success

## Cycle 1: Cloning and Transformation

### Design

---

Prior to our protein work we had to make several preparations.
The purpose of the first engineering cycle was to design the dCasMINI and sgRNA, model them to make sure the binding activity was plausible and lastly integrated the designed protein sequences into the expression strain.
For our build we will use the programs Geneious and PyMol for in silico design part.
These tools are frequently used in academia for both visualization and design of protein sequences, granting us information about the proteins that would be difficult to acquire otherwise.
Furthermore the protein we work with also is well characterized online so there are many resources available for us to use in combination with the tools.
For the wet lab part we will use E.coli TOP10 cells as the cloning strain to make sure that the designed construct is correctly integrated.
The designed construct will be assembled with constructs ordered from IDT and pASK-IBA2 vector donated by our supervisors.
Through an agarose gel we can visualize the successful integration of the construct into the vector and if the bacteria have carry them.
From this DBTL cycle we expect an expression strain with an integrated construct-plasmid with sizes as expected from the in silico design, aswell as proof that the binding of the dual casMINI complex is plausible with little steric hindrance.

### Build

---

Geneious was used for the in silico design of our dCasMINI variants and sgRNAs.
PyMol was used for the visualization of casMINI binding to make sure that there was no significance steric hindrance that may disrupt the dual binding action.

In this DBTL cycle it is quite easy to modify the build, depending on how large the changes are the team may have to redo the in silico design.

For the wetlab part, E. coli TOP10 cells were used to make two separate cultures and in these cultures a pASK-IBA2 vector with either HLC or CLS was inserted.
Therefore the wetlab lab work entails these two separate components that will be cloned.
In later cycles the plasmids will be transferred to the E. coli BL21 cells for expression of the proteins.
The DNA parts and reagents required for the wetlab were supplied by IDT and EvoGene.

The E. coli TOP10 cells have the CRISPR-CAS system endogenously which was one of the reason we used the casMINI system for our project as it is not toxic for the cells.
Therefore we don’t need to worry about stress responsed by the bacteria when expressing our genes.

In this build we used the ampicilin antibiotic to screen for the correct builds as the pASK-IBA2 vector includes ampicilin resistance.
Furthermore, during the lab we performed agarose gel to make sure we had bands of the expected size.
As a last checkpoint to make sure our construct was successfully integrated, we sent our plasmids to sanger sequencing by Eurofins Genomics.

### Test

---

Multiple tests were required for a successful engineering cycle, these tests include checking successful linearization of constructs and vector, ensuring correct integration of construct into the pASK-IBA2 vector and check transformation of the plasmid into the cloning strain E. coli TOP10.

These tests were ran simultaneously for both the HLC and CLS construct in multiple parts.

Most of these tests were done by running the construct, vector or plasmid through a agarose gel and compare the sizes to the expected size.
While throughout the experiment we also grew the colonies on cultures with antibiotics where we could observe correct transformation of the plasmid into the cloning strain as without the vector the TOP10 cells would not grow in the culture.

The results of these tests was qualitative in the form of pictures of the gels which you can access on our (link results) results.
Beside the gels there is a size ladder with known sizes that are used to compare the test to the standard sizes.
We need to make sure that the size are correct, as without the presence of these bands we cannot be sure that our experiment was successful.

### Learn

---

From this engineering cycle we wanted to learn about the optimal design of the construct, and optimalize the protocol for cloning of the construct.

The system did work as anticipated and during this engineering cycle we did learn the optimal design of the construct and the protocol for the cloning of the construct seemed to be suitable.
As the first plasmid preparation was unsatisfactory we had to make some changes to our experiment which allowed us to improve it.

Our data is displayed on the wiki which allow objective reading of our results, furthermore multiple team members analyse the data to make sure we do not incorrectly interpret the data gathered.

## Cycle 2: Protein Expression

### Design

---

The purpose of our second engineering cycle was to optimize our protein expression.
Our engineered system would optimally produce a lot of our dCasMINI protein in a soluble fraction instead of an insoluble fraction.
Proteins in the soluble fraction are easier to work with and do not require extensive lab work to use. For this engineering cycle we used E. coli BL21 (DE3) Gold cells as chassis for our expression strain.
The expression strain was built from the construct-pASK-IBA2 plasmid and inserted into the BL21 (DE3) Gold cells.
Furthermore, the dCasMINI expression function of the BL21 (DE3) Gold cells was tested in multiple conditions to check for the optimal condition.
Our experimental data will show a large band at our protein size for atleast one of the conditions. Based on the literature we do expect this protein to be soluble.

### Build

---

The engineered system was created as two separate BL21 cultures growing the His-tag-casMINI and the Strep-tag-casMINI, both cultures were grown simultaneously as they required similar experiments during the engineering cycle.
These two cultures are variants of each other as they both as based on the dCasMINI protein-cas12f but with some changes due to the presence of either a his or strep tag, for this reason it was important to keep these two separated as to not contaminate one culture with the other.
In this build it would be easy to change the protein expression given that the cloned plasmids are avaliable, if they arent avaliable some additional time will be added as we would need to synthesize new plasmids from the start.
As described in previous engineering cycle the CAS protein family is endogenous for the E. coli and by looking at avalible literture we ensured that the proteins were not toxic for the bacteria.
The BL21 (DE3) Gold cells were grown in antibiotic nutrients, which kills the bacteria that do not have antibiotic resistance granted by the pASK-IBA2 vector, furthermore we made sure to run PCRs that would confirm the presence of the construct.
This build was made with DNA produced by us in prior experiments and reagents from the EvoGene lab.

### Test

---

The most important aspects of this cycle was testing the optimal protein expression.
To determine the optimal protein expression we examined the amount of protein that was in the insoluble fraction and the soluble fraction.
After centrifugation the soluble fraction of the cells remain in the solution after lysis while the insoluble fraction form a pellet.
To further expand the soluble fraction remains soluble in the liquid and is easy to move afterwards while the insoluble fraction is hydrophobic and forms a pellet to avoid interaction with water making it difficult to work with in liquids.
The two soluble/insoluble ratio is affected by two factors, the temperature and time. E. coli has an optimal growth condition of 37 °C, however that does not mean that it is the optimal temperature for protein expression.
Therefore we test multiple temperatures to find the temperature that has the highest soluble to insoluble ratio. Furthermore the protein expression is induced by an inducer, the time spent expressing the protein also affects the ratio.
Based on this we had multiple conditions where we varied both the time and the temperature.
These tests were done with SDS-pages were we could visualize the amount of target protein in the soluble and insoluble fractions.
The results from the SDS-PAGE are qualitative. Images of the gels are periodically added to the results section as the tests are completed.
You can view them here (link to results).

To make sure that the system functions as expected we will analyse the gels and compare the postitive and negative controls with the the solube and insoluble fractions.
A large soluble fraction is the most crucial part of this engineering cycle as it allows advancement to the protein purification part.

### Learn

---

From this engineering cycle we want to learn what conditions result in the largest amount of target protein in the soluble fraction compared to the other conditions.
During the test phase of the cycle the system worked as expected and allowed detailed analysis of the optimal protein expression condtions.
However even with optimal conditions there were significant presence of the proteins in the insoluble fractions, therefore to improve the system it may be required to modify the proteins to increase solubility.
The protein was more insoluble than we expected based on the literature read in the design part.
Our tests should be presented with the most optimal condition in focus as it is the most important take away from this engineering cycle.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

#####

#### In Silico

---

Geneious Prime: A bioinformatics software platform used for sequence analysis, this enables scientist to test restriction reactions, gibson assembly, design primers and other.

Alphafold: A protein structure database that allows to troubleshoot recombinant proteins in silico. This allows to test if modified proteins fold an maintain their function.

Benchling: A bioinformatics software platform that can be used for sequence alignment in addition to other sequence analysis tools.

PyMol A molecular visualization system for molecular modeling, in our case of our CRISPR system

#####

#### PCR linearization

---

By use of primers designed to exclude a specific region in a plasmid, a plasmid can in large part be amplified and made into a linear copy.
This also produces appropriate overhangs that were designed to function with our construct G blocks.
The resulting PCR product of this linearization contains the desired linear sequence, with compatible overhangs with out G-block constructs.
After PCR to confirm that the linearization reaction had worked, a restriction digestion reaction was set up with both the PCR product and non-linearized plasmid.
A restriction endonuclease is chosen that has a restriction site in the plasmid but which is lost after linearization.
After the digestion is complete this reaction is subjected to gel electrophoresis and analyzed.

Then the PCR products were cleaned and the concentration was measured using Nanodrop to move on to Gibson assembly.

#####

#### Gibson assembly

---

This method is based on 3 enzymatic activities:

1. Exonuclease activity – creating ssDNA overrhangs for dsDNA that can then anneal to a ssDNA complement (in this case our g blocks).
2. DNA polymerase activity – for filling in the gaps after annealing of our ssDNA and the g blocks.
3. DNA ligase activity – covalently sealing nicks in our assembly.

It’s an efficient way to join and clone DNA molecules larger than 300 kb into cloning vectors (in our case pASK-IBA2). These constructs were sequence verified and then cloned.

#####

#### Digestion with BamHI

---

BamHI – restriction endonuclease, was used to digest the circular pASK-IBA2 vector as a control. The linearized pASK-IBA vector has lost the BamHI restriction site.

#####

#### Transformation of TOP10

---

After fragments and linearized vector (pASK-IBA2) were subjected to Gibson Assembly Master Mix and incubate at 50°C for 15 minutes to 1 hour were transformed into TOP10 cells.
TOP10 cells are useful for clonning due to their high transformation efficiency [1].

#####

#### Transformation of E.coli

---

Heatshock transformation is a method used to introduce foreign DNA into bacteria.
In our case we introduced pASK-IBA2 vector with our constructs: HLC and CLS.
BL21α cells were transformed in order to start protein expression experiments.
BL21α are used for expression for non-toxic heterologous genes and is an efficient expression strain [2].

#####

#### Small-scale expression experiments

---

Small-scale expression was conducted in order to optimize protein expression conditions.

We decided to test our construct expression in 16°C, 30°C, 37°C and room temperature (22-24°C) and inducing the expresion after 1 h, 3h and over night.

#####

#### Large-scale expression experiments

---

Large-scale expression was conducted after optimization (30°C degrees overnight induction) to move on to purification.

#####

#### His-tag purification

---

Recombinant HLC protein has 6 Histidine residues chosen to purify the construct under Ni-NTA affinity purification of a bind-wash-elute procedure.

N-terminal His-tagged protein (HLC) was purified on a nickel-nitrilotriacetic acid (Ni-NTA) HisTrap™ FF 1 ml column (GE Healthcare) with a sepharose matrix pre-charged with nickel ions.
The column was equilibrated in 5 column volumes (CV) buffer A (see buffer recipes – his-tag purification buffer a). Before the filtered supernatant oF HLC wasloaded onto the column using a 50 ml superloop (GE Healthcare).
The column was then washed with 10 CV buffer a (see buffer recipes – his-tag purification buffer a) before HLC was eluted during a 0-100% gradient of buffer b (see buffer recipes – his-tag purification buffer b) over 15 CV.

Fractions from the chromatography are analysed on an sds gel.

#####

#### Strep-tag purification

---

A Strep-tag is a synthetic peptide (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and was fused to our protein for efficient purification using an affinity column.

Bis-Tris Native gel [3]

The native gel system we used has a near neutral pH (~7.5) to perform native (non-denaturing) electrophoresis. This environment results in better band resolution due to the stability of the protein and the gel matrix.

The NativePAGE ™ Gel system is based on the Blue Native Polyacrylamide Gel Electrophoresis (BN PAGE) technique developed by Schägger and von Jagow (Schägger & von Jagow, 1991) that uses Coomassie G-250 as a charge-shift molecule [1].
After binding to the proteins the charge shift molecule confers a negative charge but does not denature the protein – leaving it in its native state.
The Coomassie G-250 is in the cathode solution that is applied to the gel continuasly.

#####

#### SDS PAGE

---

Is a method to separate proteins based on their size, all the proteins in a complex mixture are denatured and have a negative charge and they are separated by size while they move towards the positive probe.

Agarose electrophoresis

Gel electrophoresis method used to separate macromolecules such as DNA, RNA and protein in an agarose matrix. Proteins can be seperated by their isolelectric charge (not applied in our project), DNA and RNA can be seperated by length.

Gel-Red staining

An intercalating molecule that when bound to nucleic acid and exposed to UV light strongly fluoresceces. It is safer due to being less toxic than ethidium bromide.

Silver staining

A staining method that has a sensitivity in the nanogram range. The working principle is silver ions binding to the protein that are then reduced under appropriate conditions that results in a visible image from a fine coat of silver.

Analytical chromatography

Since we did not succeed in visualizing our CasMINI proteins binding the RNA with native gel or agarose gels we decided to run our samples through an analytical column and overlap the resulting chromatograms.
In theory, gRNA only sample, CasMINI only sample and gRNA bound to CasMINI sample should have different sizes, which is reflected in chromatograms after running them through the column.

## References

1. https://www.thermofisher.com/no/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-strains/top10-competent-cells.html
2. https://www.thermofisher.com/order/catalog/product/EC0114
3. https://tools.thermofisher.com/content/sfs/manuals/nativepage\_man.pdf

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

To build our detection system we applied a plethora of methods and kits.
Among others our team had to optimize expression conditions, use different purification columns, staining methods and optimize CasMINI-gRNA binding conditions.
Not all applied methods gave successful results, however, we were still able to achieve positive results in the end.
To make interpretation of our results easier we introduce abbreviations used in annotations and notes:

- HLC (His-L-Cas) – stands for the construct of dCasMINI with an N terminally linked His-tag.
- CLS (Cas-L-Strep) – stands for the construct of dCasMINI with a C terminally linked strep-tag.
- gBlocks – gene fragments that are double stranded DNA sequences 125–3000 bp in length.
- ON or O/N - overnight, referred in expression experiments for the length of the expression (~17 h)
- RT – room temperature (~22°C)
- sgRNA – guide RNA, engineered to bind to a DNA sequence transcribed from a potential endometriosis microRNA biomarker miRNA199b (1)
- SDS-PAGE: Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis. Novex WedgeWell 4%-20% Tris Lysine gel (Invitrogen). See protocols for details.

---

## Cloning and Transformation

##### *Aim*

- Insert our CasMINI coding sequence into pASK-IBA2 a commonly used expression vector.
- Transform our cloning strain (TOP10) with our plasmids.
- Identify and subclone plasmids containing successfully inserted genes into BL21 (DE3) Gold expression strain.

##### *Conclusion*

- Using the Gibson Assembly method, we successfully inserted our G-blocks coding for the two dCasMINIs into the pASK-IBA2 vector.
- The E.coli Top10 cloning strain was successfully transformed with our plasmids containing our respective genes.
- The E.coli BL21 (DE3) Gold expression strain was successfully transformed with our plasmids containing their respective genes.

After **isolating** plasmid of interest (pASK-IBA2) from *E. coli* DH5α, we linearized it by PCR using appropriate primers.

**Gibson assembly** (see “[Gibson assembly](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D)” in protocols under cloning and transformation) was performed to insert our construct coding-sequences into the linearized vector.
We succeeded in creating HLC and CLS coding-sequence-containing plasmids.

*E.coli* Top10 cells were **transformed** with our plasmids, and **single-colony PCR** with primers targeting insertion site was performed at the same time as colonies were inoculated in liquid culture.
The gel shown in Figures 1 and 2 indicates the successful insertion of gBlock sequences, as seen by the bands corresponding to ̴ 1500 kB.
The results of the gels were later confirmed by **Sanger sequencing** of the isolated plasmids.

![Gel1](https://static.igem.wiki/teams/4899/wiki/images-results/results-figure-1.png)

**Figure 1:** 1% agarose gel showing bands obtained from gel electrophoresis (100 Volt, 30 minutes).
Lane 1 contains the 0`GeneRuler 1 kB ladder;
Lanes 2-9 contain the chosen **TOP10** colonies to be tested for the HLC. For more details, head over to our
[protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) &
[notebook.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

![Gel2](https://static.igem.wiki/teams/4899/wiki/images-results/results-figure-2.png)

**Figure 2:** 1% agarose gel showing bands obtained from gel electrophoresis (100 Volt, 30 minutes).
Lane 1 contains the 0`GeneRuler 1 kB ladder;
Lanes 2-8 contain the chosen **TOP10** CLS colonies. For more details, head over to our
[protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) &
[notebook.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

*E.coli* BL21 (DE3) Gold cells were then transformed with the plasmids which had no mutations deemed to be harmful to expression.
After transformation single colony PCR was performed, the result of which is seen in Figure 3 where bands of expected size ̴ 1500 kB are present.
The bands seen in figure 3 (wells 2 through 9, with well 1 containing the ladder) seem to deviate slightly in size, this could potentially have been caused by the gel itself not being perfectly cast.

![Gel3](https://static.igem.wiki/teams/4899/wiki/images-results/results-figure-3.png)

**Figure 3:** 1% agarose gel showing bands obtained from gel electrophoresis (100 Volt, 30 minutes) of a single colony PCR of **BL21 (DE3) Gold cells**.
Lane 1 contains the 0`GeneRuler 1 kB ladder;
Lanes 2-5 contain the chosen HLC colonies, lanes 6 to 9 contain the chosen CLS colonies. For more details, head over to our
[protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) &
[notebook.](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)

One of each colony for HLC and CLS were chosen. Glycerol stocks were made and kept at -80°C for future experiments.

## Protein Expression

### Small Scale Protein Expression (CLS)

---

##### *Aim*

- Confirming successful transformation of our CLS construct in E.coli BL21(DE3) Gold
- Optimizing expression conditions for our CLS construct in E.coli BL21(DE3) Gold

##### *Conclusion*

- - pASK-IBA2 vector with a CLS insert was transformed into E.coli BL21(DE3) Gold, since we see a band ~64 kDa (CLS protein size).
  Optimal expression conditions are room temperature (~22°C)

After confirming that BL21 single colonies were transformed with our plasmids (se figure 3) we continued with small-scale expression to figure out optimal conditions.
At first, we noticed that most of our protein ended up in the insoluble fraction (see figure 4).

![Gel4](https://static.igem.wiki/teams/4899/wiki/small-scale-hlc-cls-37-3.png)

**Figure 4:** 4-20% SDS-PAGE gel image of CLS expression. Induction at 37°C for 3h.
Mechanical lysis with bead beater.
Lane 1 contains the ladder - PageRuler™ Plus Prestained protein Ladder 10-250 kDa, lanes 2-5 contain HLC samples, and CLS samples are in lanes 6-9.

Based on articles...

cont...

![Gel5](https://static.igem.wiki/teams/4899/wiki/small-scale-cls-16.png)

**Figure 5:** 4-20% SDS-PAGE gel image of CLS expression. Induction at 16°C for 1h, 4 h and overnight.
Mechanical lysis with bead beater.
Lane 1 contains the ladder - PageRuler™ Plus Prestained protein Ladder 10-250 kDa, lanes 2-5 contain 1 h induction samples, 6-9 4-hour induction samples and 10-15 o/n samples of CLS.

BL21 (DE3) Gold cells were then transformed with the plasmids which had no or few mutations deemed to be harmful to expression. After transformation single colony PCR was performed, the result of which is seen in Figure 3 where bands of expected size ̴ 1500 kB are present. The bands seen in figure 3 (wells 2 through 9, with well 1 containing the ladder) seem to deviate slightly in size, this could potentially have been caused by the gel itself not being perfectly cast.

![Gel6](https://static.igem.wiki/teams/4899/wiki/small-scale-cls-rt.png)

**Figure 6:** SDS-PAGE gel image of CLS expression. Induction at RT for 1h, 4 h and overnight.
Mechanical lysis with bead beater.
Lane 1 contains the ladder, lanes 2-5 contain 1 h induction samples, 6-9 4-hour induction samples and 10-15 o/n samples of CLS.

##### *Sample Descriptions*

Unind. = Uninduced sample, whole cell
Ind. = Induced sample, whole cell
Insol. = Insoluble sample, cell lysate
Sol. = Soluble sample, cell lysate

![Gel5](https://static.igem.wiki/teams/4899/wiki/small-scale-cls-30.png)

**Figure 7:** 4-20% SDS-PAGE gel image of CLS expression. Induction at 30°C for 1h, 4 h and overnight.
Mechanical lysis with bead beater.
Lane 1 contains the ladder - PageRuler™ Plus Prestained protein Ladder 10-250 kDa, lanes 2-5 contain 1 h induction samples, 6-9 4-hour induction samples and 10-15 o/n samples of CLS.

![Gel5](https://static.igem.wiki/teams/4899/wiki/small-scale-cls-37.png)

**Figure 8:** 4-20% SDS-PAGE gel image of CLS expression. Induction at 37°C for 1h, 4 h and overnight.
Mechanical lysis with bead beater.
Lane 1 contains the ladder - PageRuler™ Plus Prestained protein Ladder 10-250 kDa, lanes 2-5 contain 1 h induction samples, 6-9 4-hour induction samples and 10-15 o/n samples of CLS.

### Small scale expression experiments (HLC)

---

##### *Aim*

- Confirming successful transformation of our HLC construct into E.Coli BL21 (DE3) Gold expression strain
- Optimizing expression conditions for our HLC construct in E.Coli BL21 (DE3) Gold

##### *Conclusion*

- pASK-IBA2 vector with a HLC insert was transformed into E.Coli BL21 (DE3) Gold, since we see a band ~64 kDa (HLC protein size).
- Optimal expression conditions are 16°C with an induction time of 17 hours.

After confirming that BL21 single colonies were transformed with our plasmids (se figure 3) we continued with small scale expression to figure out optimal conditions.
At first, we noticed that most of our protein ended up in the insoluble fraction (figure 9).

Due to both HLC and CLS being tagged variants of dCasMINI (a modified cas12f), we decided to test 16 °C overnight and room temperature both of which gave large amounts of CLS in the soluble fraction.
We decided to test if the different tags between the 2 proteins would affect their optimal expression condition.
After testing both conditions, we determined that the highest amount of HLC in the soluble fraction was 16 °C overnight (figure 10).

![Gel4](https://static.igem.wiki/teams/4899/wiki/bb-hlc-cls.png)

**Figure 9:** 4-20% SDS-PAGE gel image of HLC expression. Induction at 37°C for 3h.
Mechanical lysis with bead beater.
Lane 1 contains the ladder - PageRuler™ Plus Prestained protein Ladder 10-250 kDa, lanes 2-5 contain HLC samples, and CLS samples are in lanes 6-9

![Gel5](https://static.igem.wiki/teams/4899/wiki/hlc-expression-testing-16c-rt.png)

**Figure 10:** 4-20% SDS-PAGE gel image of HLC expression induction for overnight in 16°C and room temperature.
Mechanical lysis with bead beater.
Lane 1 contains the ladder - PageRuler™ Plus Prestained protein Ladder 10-250 kDa, lanes 2 to 5 contains 16 °C overnight induced samples, and lanes 6 to 9 contains room temperature (22 °C) overnight induced samples.

## Protein Purification

### CLS: Purification of our strep-tagged construct

---

##### *Aim*

- To test if increasing the culture volume still allowed for efficient expression of our construct.
- To produce large amounts of protein that could be purified (using Strep-tag purification) and used in following binding assays.

##### *Conclusion*

- The conditions that were found to be optimal for small-scale expression of our CLS construct held true for large-scale expression.
- Satisfactory amounts of our construct was purified to be used in further experiments.

After successfully producing CLS on a larger scale (2x1L) we aimed to purify the protein using a StrepTrap XT column packed with Strep-Tactin® XT Sepharose resin to purify our Strep-tagged CLS protein [3].
The chromatogram in figure 11 shows a single large peak after the isocratic point which indicates the addition of buffer e (see buffers).
For affinity chromatography with this specific column, this is likely to be the point where our target protein was eluted.
The rather slow drop in the UV signal indicates that not all of the CLS protein was eluted simultaneously and that there are other proteins that were bound to the column. This was confirmed by SDS gel of the fractions produced (see figure 12).

![Gel4](https://static.igem.wiki/teams/4899/wiki/chromatogram-cls.png)

**Figure 11:** Chromatogram of CLS after Strep tag purification conducted with ÄKTA start protein purification system, using a StrepTrap XT 1ml column.

![Gel5](#)

**Figure 12:** 4-20% SDS-PAGE gel of fractions collected from his-tag purificaiton of CLS construct. The first lane contains the PageRuler™ Plus Prestained protein Ladder 10-250.
The annotation in the figure of F# indicates which fraction the lane contains a sample from.
Large amounts of protein are present in lanes F11 to F15, most of the purified sample is present in F11 to F13.

Several fractions were run on a gel to confirm which fractions could be combined and further concentrated.
Figure 12 shows that fractions F11 to F14 could be combined and concentrated with a 20 ml ~30 kDa molecular weight cut off filtration filter tube (vivaspin centrifugal concentrators).
The end protein sample concentration was 39.6 µM. This is enough for a stock amount to then later be used in EMSA – protein/sgRNA binding assays. As can be seen in figure 13 the band corresponding to ~64 kDa has decreased in proportional size and intensity, whilst multiple other bands, particularly smaller bands are gone or weaker relative to the ~64 kDa band.
The presence of smaller bands indicates that either CLS is being degraded or that the concentration was done improperly.
Whilst the decrease in the intensity of the 64 kDa can be due to limitations in the taken sample or improper concentration.

![Gel6](https://static.igem.wiki/teams/4899/wiki/cls-concentrated.png)

**Figure 13:** 4-20% SDS-PAGE gel of unconcentrated CLS and concentrated CLS after purification.
1 lane - PageRuler™ Plus Prestained protein Ladder 10-250 kDa,
2 lane – unconcentrated CLS,
3 lane – concentrated CLS

### HLC: Purification of our His-tagged construct

---

##### *Aim*

- To test if increasing the culture-volume still allowed for efficient expression of our construct.
- To produce large amounts of protein that could be purified (using His-tag purification) and used in following binding assays

##### *Conclusion*

- The conditions that were found to be optimal for small-scale expression of our HLC construct held true for large-scale expression.
- Satisfactory amounts of our construct was purified to be used in further experiments.

After successfully producing HLC on a larger scale (2x1L cultures) we aimed to purify the protein using a HisTag FF column.
The chromatography results of the His-tag purification show one peak (figure 14) that likely corresponds to eluted HLC.
To not lose most protein and confirm which fractions contain HLC we ran an SDS-PAGE gel. According to figure 15 fractions 9 to 13 were eligible to be combined and concentrated, which resulted in 22.22 µM of HLC.
This is enough stock amount to have later be used in EMSA – protein/sgRNA binding assays and protein/sgRNA/DNA binding assays.

For affinity chromatography with this specific column, this is likely to be the point where our target protein was eluted.
The rather slow drop in the UV signal indicates that not all of the CLS was eluted simultaneously and that there are other proteins that were bound to the column.
This was confirmed by SDS gel of the fractions produced (figure 15).

![Gel4](https://static.igem.wiki/teams/4899/wiki/chromatogram-hlc.png)

**Figure 14:** Chromatogram of HLC purification with a HisTag FF column results for HLC, using HisTrap FF, 1 ml column.
Conducted with ÄKTA start protein purification system.

![Gel5](https://static.igem.wiki/teams/4899/wiki/his-tag-purification.png)

**Figure 15:** 4-20% SDS-PAGE gel of fractions collected from his-tag purificaiton of HLC construct.
The first lane contains the PageRuler™ Plus Prestained protein Ladder 10-250. Lanes 2 and 3 contains samples collected before the culture was pelleted and samples take from the supernatant during loading on the affinity column after dilution.
The annotation in the figure of F# indicates which fraction the lane contains a sample from.
Of the fractions most of the proteins in the sample are in F3.
However the largest amount of purified protein are present in lanes F11 to F15, with most of the purified sample being in F11.

Several fractions were run on a gel to confirm which fractions could be combined and further concentrated.
Figure 15 shows that fractions F9 to F13 could be combined and concentrated with a 20 ml ~30 kDa molecular weight cut off filtration filter tube (vivaspin centrifugal concentrators).
The end protein sample concentration was 22.22 µM. This is enough for a stock amount to then later be used in EMSA – protein/sgRNA binding assays.
As can be seen in figure 16 the band corresponding to ~64 kDa has increased in proportional size and multiple other bands, particularly smaller bands are gone or weaker relative to the ~64 kDa band.
The presence of smaller bands indicates that either HLC is being degraded or that the concentration was done improperly .

![Gel4](https://static.igem.wiki/teams/4899/wiki/hlc-concentrated.png)

**Figure 16:** 4-20% SDS-PAGE gel electrophoresis of unconcentrated HLC and concentrated HLC after purification.
1 lane – ladder - PageRuler™ Plus Prestained protein Ladder 10-250 kDa,
2 lane – I unconcentrated HLC, fraction 11;
3 lane – II conmbined and concentrated HLC.

## Transcription of sgRNA

### Transcription of sgRNA

---

##### *Aim*

- Produce sgRNA of functional quality for following EMSA (electrophoretic mobility shift assay) experiments.

##### *Conclusion*

- sgRNA of both variants could be transcribed in vitro with sufficient yield.
- Both sgRNAs were of sufficient quality to be used in later EMSA experiments.

sgRNA synthesis was done using the New England BioLabs T7 hiscribe in vitro transcription kit according to the Standard RNA Synthesis (E2040) protocol (new England biolabs).
The reaction was incubated for 4 hours before the RNA was treated with DNase 1 (DNase1 see protocols), and the reaction was purified in a Qiagen RNeasy clean-up mini kit.
The resulting RNA sample was then analyzed with a nanodrop to estimate concentration, and with a bioanalyser using a total RNA nano kit to estimate RNA quality.

The sgRNA1 gave a concentration of 35,4 ng/ul and showed some peaks for shorter than the intended sgRNA size during bioanalyzer analysis (se figure 15).

![sgrna1](https://static.igem.wiki/teams/4899/wiki/sgrna1.png)

**Figure 17:** This figure shows the result of analysis with a bioanalyzer of sgRNA1 the peaks furthest to the right (marked 188nt) is near the expected size of the sgRNA1.
The size of this peak compared with the others indicates that the quality of the RNA sample is usable for our purpose.

The sgRNA2 gave a concentration of 33,2 ng/ul and showed some peaks for shorter than the intended sgRNA size during bioanalyzer analysis (figure 16).

![sgrna2](https://static.igem.wiki/teams/4899/wiki/sgrna2.png)

**Figure 18:** this figure shows the result of analysis with a bioanalyzer of sgRNA2 the peaks furthest to the right (marked 188nt) is near the expected size of the sgRNA2.
The size of this peak compared with the others indicates that the quality of the RNA sample is usable for our purpose.

## CasMINI and sgRNA Binding Experiment

### CasMINI and sgRNA Binding Experiment

---

##### *Aim*

- To find the optimal conditions for the binding of the dCasMINI proteins to the sgRNAs

##### *Conclusion*

- Current buffers and/or conditions do not facilitate the binding of the sgRNA and our dCasMINIs.

To test the binding of our sgRNAs to the dCasMINI proteins, we decided to perform an EMSA (electrophoretic mobility shift assays) experiment which would allow easy visualization of the binding by the change in band position for our RNA and/or protein (depending on the staining method).
As Xu et.al only worked with cell cultures, and our test intended to be done in an in vitro non-cellular environment. We found it necessary to establish the optimal conditions for this reaction.
As such we based our original method on the work of Li et.al who worked with the structure of “apo Lb2Cas12a”, a cas protein in the same family as Cas12f (and by extension dCasMINI).
The reaction was conducted in the binding buffer (link here to RNA binding buffer), at 4°C for 15 minutes before being loaded on a gel.

First, we tried running Native gels to visualize our protein constructs binding to the sgRNA.
Due to the sgRNA DNA template primers being delivered late, we had to work with a very small amount of our sgRNA. This influenced our reaction volume.
We decided to work with 100 nM sgRNA with 100 nM and 200 nM of protein in a 5 ul reaction.
The total amount of protein in this reaction was not sufficient for the staining methods attempted (se figure11) for visualization of the protein.

![Gel4](https://static.igem.wiki/teams/4899/wiki/native-cls-dilutions.png)

**Figure 19:** 8% to 16% Native-page Bis-Tris Gel electrophoresis EMSA – CLS and sgRNA binding and serial dilutions of CLS, silver staining.
Annotations: I – sgRNA 100 nM, II – gRNA 100 nM + CLS 100 nM; III - gRNA 100 nM + CLS 200 nM; V – stock of CLS – 11.11 µM; VI – 5.5 µM CLS; VII – 0.5 µM CLS; VIII – 200 nM CLS; IX – 100 nM CLS.
There seems to be some spillover from the loading of the stock protein solution as seen by the large band present in all lanes, but weakening away from the start of the serial dilution.
First lane - PageRuler™ Plus Prestained protein Ladder 10-250 kDa

We then decided to run a 2% agarose gel electrophoresis like that done in the protein RNA interaction work of Ream et.al.
When the reaction was run on an agarose gel we were able to see weak bands for RNA in the expected size.
This proved to us that dcasMINI did not bind to sgRNA under these conditions, since RNA bands were visible in all 3 loaded wells at the same length (Figure 20).
This also demonstrated that our RNA was not degraded under these conditions.

![Gel5](https://static.igem.wiki/teams/4899/wiki/agarose-rna.png)

**Figure 20:** 2% agarose gel 100V 50 min.
I – gRNA without protein, control;
II – 100 nM sgRNA incubated with 100 nM CLS for 15 min in 4°C;
III – 100 nM gRNA incubated with 200 nM CLS for 15 min in 4°C. 2% agarose gel 70V 50 min.
I – gRNA without protein, control;
II – 100 nM gRNA incubated with 100 nM CLS for 15 min in 4°C;
III – 100 nM gRNA incubated with 200 nM CLS for 15 min in 4°C.

We saw no binding in the starting conditions we decided to try to optimize.
Seeing that Kupcinskaite et.al and Anders et.al used higher temperatures and longer reaction times, we decided to increase the incubation temperature and duration for our reaction. We then tested incubation at 37 °C for 25 minutes.
The reaction was loaded on a 2% agarose gel to visualize a change in the migration of the sgRNA bands. Unfortunately, these changed conditions did not cause any binding to occur.
This is seen by the lack of any bands of greater size than those seen in the control (see fig), a band of greater size would indicate that the RNA has been bound to something that slows its progress through the gel.

![Gel6](https://static.igem.wiki/teams/4899/wiki/figure-21-37c-25-min.jpg)

**Figure 21:** 2% agarose gel 70V 50 min.
I – gRNA without protein, control;
II – 100 nM gRNA incubated with 200 nM CLS for 25 min in 37°C.

## Experiments that did not succeed yet:

We have yet to optimize binding conditions for our guide RNAs and CasMINI proteins.

## Future plans:

- Optimize sgRNA and CasMINI binding conditions
- Develop different guide RNAs to detect an array of potential early endometriosis biomarkers
- Develop a physical lateral flow test

## References

1. Zafari, N., Tarafdari, A.M., Izadi, P. et al. A Panel of Plasma miRNAs 199b-3p, 224-5p and Let-7d-3p as Non-Invasive Diagnostic Biomarkers for Endometriosis. Reprod. Sci. 28, 991–999 (2021). https://doi.org/10.1007/s43032-020-00415-z
2. Chapron C, Marcellin L, Borghese B, Santulli P. Rethinking mechanisms, diagnosis and management of endometriosis. *Nat Rev Endocrinol*. 2019 Nov;15(11):666-682. doi: 10.1038/s41574-019-0245-z. Epub 2019 Sep 5. PMID: 31488888.
3. Ringen, I.M. (2021) Endometriose. Available at: <https://legehandboka.no/handboken/kliniske-kapitler/gynekologi/tilstander-og-sykdommer/menstruasjonsproblemer/endometriose> (Accessed: 24.07.2023).

{% endblock %}