iGEM 2024 — Team uioslo-norway

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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# Notebook

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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# Engineering Cycle 01 - The Plasmid and E. coli

![1-Engineering](https://static.igem.wiki/teams/5333/engineering/engineering-cycle-1.png)

## During this engineering cycle, the sequence used for cloning was prepared, and transformation of the expression strain conducted.

### Design

The purpose of our design is to express the glucocorticoid receptor (GR) with a His-tag for purification from Escherichia coli cells. The GR must be prepared for expression from the vector including a his-tag for purification and attachment to an electrode for later ligand-binding measurements. The prepared sequence is then used to transform the expression strain.

The sequence for the design is retrieved from online databases such as UniProt and the RCSB Protein Data Bank. The protocols by He et al. 2014 are used as guidance for the build.

In the wet lab for cloning the construct Top10 E.coli competent cells are used. The G-block sequence pET-28-His-GR is ordered from Integrated DNA Technologies (Ref# 239413871) and cloned by Gibson Assembly into the linearised pET-28a(+) vector, which is from Novagen, carries as kanamycin resistance gene, requires as T7 polymerase for expression, and has a thrombin cleavage site right after an n-terminal his-tag. With Colony PCR and Gradient PCR the successful incorporation of the G-block into the vector is verified.

In this engineering cycle we prepare the plasmid used for expression, transform cells to an expression strain, and verify the successful cloning to continue expression and purification of GR.

### Build

The GR must be prepared for expression in a plasmid and with the His-tag right after the thrombin cleavage site (Simmons et al. 2008). The sequence can be found in the RCSB Protein Data Bank and the protocols are from He et al. 2016.

The sequence was then prepared by identifying the required LBD with the Bledsoe et al. 2002 publication, introducing mutations that are improving solubility; F602A, C622Y, T668V, S674T, V675I (He et al. 2014) and adding the His-tag based on Simmons et al. 2008 and He et al. 2014 protocols. From the European Nucleotide Archive by EMBL-EBI (entry X03225.1) the mRNA sequence for the protein of interest was retrieved. After rare codon analysis with GenScript Rare Codon Analysis Tool and codon optimisation for expression in E. coli with Sequence Optimizer by Twist Bioscience of the region of interest, flanking sites for introduction into the pET-28a(+) vector were added. The complete sequence is called the G-block.

In the wet lab experiments the pET-28a(+) vector and G-Block were inserted into the Top10 competent E.coli culture. Later on the plasmids were transferred to a protein expression strain of BL21(DE3) Competent E. coli. The components for the cloning procedure were the G-Block plasmid prepared by us and produced by Integrated DNA Technologies, and reagents provided by the facilities of the EvoGene research division at the University of Oslo.

The bacteria was cultured with kanamycin as a selection marker for successful transformation as the pET-28a(+) plasmid contains a kanamycin resistance gene. Additionally, Colony PCR and Gradient PCR were performed to verify correct cloning resulting in successful plasmid preparation.

### Test

For a successful engineering cycle, the linearisation of constructs, vector, and proper integration of the G-block into the pET-28a(+) vector must be verified, as well as proper transformation of Top10 competent E.coli.

A first indicator for correct assembly of the vector and G-Block was the survival of cells in kanamycin culture. However, to exclude the possibility of random mutations towards antibiotic resistance other verification experiments were conducted. Using Colony PCR, Gradient PCR, enzyme digestion, and agarose gel analysis, the correct band size for the plasmid was visible. The results are captured as pictures. (see [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D))

### Learn

The learning outcome aimed for in this cycle was the preparation of plasmids and all required components for said plasmid construct. Due to the essential information from He et al. 2014 about sequence optimisation, the plasmid preparation went successful and the experimental procedure could be continued with expression and purification of the GR. The results are analyzed by the team with guidance from the team’s supervisors.

# Engineering Cycle 02 - The GR Expression and Purification

![2-Engineering](https://static.igem.wiki/teams/5333/engineering/engineering-cycle-2.png)

## During this engineering cycle, the GR was expressed by E. coli and purified for further experimentation.

### Design

In this engineering cycle, the GR is expressed and purified. The resulting protein may be found in the soluble, intermediate, or solid fraction and will be investigated by using all of them separately. The expression strain of this engineering cycle is the BL21(DE3) Competent E. coli cells that have been transformed with the pET28-His-GR plasmid construct prepared with pET-28a(+) as the vector, and the G-Block carrying the GR LBD sequence with His-tag and flanking sites for insertion into the vector. The protein isolation samples are tested for different conditions to find the best suitable fraction for GR purification.

### Build

The BL21(DE3) Competent E. coli cells were cultured in kanamycin-containing broth to remove any bacteria without the resistance gene that is provided by the pET28-His-GR plasmid construct. During the first engineering cycle, the existence of the correct plasmid was verified with PCRs and agarose gel electrophoresis. Following the cultivation, the cell samples were prepared with DNAse and lysed to gather the protein. With careful centrifugation with nickel beads to bind the protein, first a soluble phase from the supernatant and a solid protein fraction from the pellet were collected. Lastly, the remaining supernatant was processed with syringe-passing for filtration. The three phases were next tested with SDS PAGE to detect GR.

The components for this lab procedure revolving GR expression were the DNA-plasmid previously prepared by us (see 1st engineering cycle) and reagents available for research use from the facilities of the EvoGene research division at the University of Oslo.

### Test

It was essential to determine the purification step that yields GR most efficiently. This was done by comparing the protein samples gathered from the supernatant (soluble protein), the pellet (solid phase protein/insoluble), and the syringe-filtered phase by SDS PAGE. The outcome of this experimental procedure did not show protein yields as expected, as they were generally too low for further experimentation (see [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)). These results urged for troubleshooting.

To improve the protein purification outcome, a different method was used. With a chromatographic separation procedure, three phases were obtained: a flowthrough phase, a washing phase, and an elution phase. In chromatographic experiments, the components in the sample are separated due to their different affinities to the stationary phase. Flowthrough samples have a very low binding affinity, while the elution phase samples have a high affinity.

Following the purification by chromatography, a concentration procedure of the samples was done to increase the amount of protein per volume, as it was too low for SDS PAGE previously.

GR is expected to be observed in the elution phase. SDS PAGE followed by Western Blotting was conducted as verification for the experiment. As expected, the protein yield increased, with the GR being found in the elution phase (see [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)).

To verify the results as correct, the different phases containing protein are compared with positive and negative controls. For further experimentation on the overall project, a proper quantity of the elution phase containing GR is essential.

### Learn

From this second engineering cycle, the adjustment of protocols towards a better protein yield and finding the correct phase for the protein of interest was the learning outcome. In the test phase, it was shown that nickel bead purification was unsuited for GR. However, chromatographic separation followed by a procedure to increase concentration resulted in evidence of GR in the expected elution phase sample on the Western Blot. This realisation is crucial for further experimentation and optimisation. However, since the protein concentration needed to be increased before analysis, optimisation of protein expression targeting the induction in the E. coli would be a natural further improvement of the experimental protocols.

## References

Bledsoe, R. K., et al., 2002. 'Crystal structure of the glucocorticoid receptor ligand binding domain...', Cell, 110: 93-105.

He, Y. Z., et al., 2014. 'Structures and mechanism for the design of highly potent glucocorticoids', Cell Research, 24: 713-26.

Simmons, C. A., et al., 2008. 'Expression, purification, and characterization of multiple, multifunctional human glucocorticoid receptor proteins', Protein Expr Purif, 62: 29-35.

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# Experiments

* [Design of GBlock (In Silico)](#design-gblock)
* [PCR linearization of vector and cloning](#pcr-lin)
* [Gibson Assembly (Insertion of GBlock)](#gibson-a)
* [Transformation of BL21(DE3)](#bl21)
* [Culture preparation and induction](#culture-prep)
* [Protein purification](#protein-pur)
* [Western blot](#western-blot)
* [Future perspectives](#future-perspectives)

## Design of GBlock (In Silico)

**Uniprot:** The Glucocorticoid Receptor ligand binding domain (GR-LBD) was identified under the entry number P04150-GCR\_Human, gene NR3C1.

**RCSB-PDB:**The specific mutations described in He, Y et al., (2014) was retrieved from Xstal structure with the entry number 4P6X.

**GenScript:** Used for rare codon analysis.

**Twist Bioscience:** Used for the codon optimization tool.

## PCR linearization of vector and cloning

A plasmid can be amplified by using PCR specific primers designed to exclude a specific region and, in this way, we will obtain a linear copy of the cloning vector to use, pET pET-28a transformed into TOP10 competent E. coli strains. The exclusion of this section of the vector produces overhangs that were considered in the design of our gBlock, and that way include our desired coding sequence into the cloning vector pET-28a.

The resulting PCR linearization products contains the coding sequence of our protein of interest...Read more content to avoid excessive length.

The final PCR products of the linearization were cleaned, the concentration and quality measured using Nanodrop. The PCR products were then prepared for the Gibson assembly.

## Gibson Assembly (Insertion of GBlock)

Gibson assembly was utilized to clone the gBlock, containing GR-LBD and His-tag, into the linearized vector obtained in the PCR linearization step. The resulting circularized vector was then to be used further to transform competent BL21(DE3) expression E. coli bacteria strain.

The Gibson Assembly master mix is based on three different enzymes withing a single buffer mixture to improve accuracy and efficiency. The enzymatic activities consist of:

* **Exonuclease activity:** Creates single-strand DNA overhangs from the linearized double stranded cloning plasmid pER-28a that can be anneal to complementary single-stranded complementary overhangs from our gBlock.
* **DNA polymerase activity:** Incorporates nucleotides to “fill” the gaps after the single strand DNA and our gBlock has been annealed.
* **Taq DNA activity:** Covalently joins the annealed complementary DNA fragments, removing any nicks and creating a contiguous DNA fragment between the cloning vector and our gBlock.

The constructs were verified by sequencing and then were transformed into Top10 E. coli competent cells to be cloned.

Colony PCR and Gradient PCR was also performed into the resulting cloned cultures to confirm that we successfully inserted our gBlock into the cloning strains.

## Transformation of BL21(DE3)

Bacterial transformation is used in molecular cloning to produce multiple copies of a DNA molecule, in our case the plasmid carrying our gBlock, and its subsequent expression. For our experiment, we choose BL21(DE3) competent E. coli strain, as it is a suitable and strain for efficient protein expression.

We use Heatshock transformation method to introduce the cloning vector with our gBlock into the expression E. coli strain. After the induced stress, the cells were incubated at 37°C in an SOC recovery medium.

## Culture preparation and induction

Small scale induction

We produce a small-scale culture to test the expression conditions of our construct and to decide what is the most optimal condition for protein production. The pre-culture corresponded 3 ml of LB-Kanamycin-50 inoculated with a single colony of our construct BL21DE3-pET28-GR. After overnight incubation at 37°C, a 1:100 dilution was done to control the growing process of the liquid culture. We decided to test the construct incubating the culture at 20°C and 37°C and inducing expression (By adding ITPG and Prednisolone) after incubations times of 30 minutes, hour hours and overnight.

Large Scale induction

The optimal conditions for production of our construct were determined on an incubation of 20°C overnight incubation. We produced a large-scale culture by making a pre-culture corresponding of 15 ml of LB-Kanamycin-50 inoculated with a single colony of our construct BL21DE3-pET28-GR. After overnight incubation at 37°C, a 1:100 dilution was done by adding 15 ml of the overnight culture in a 1 liter of LB-Kanamycin and incubating at 37°C until the ideal OD was obtained (Range between 0.4 and 0.7). The induced culture was then incubated at 20°C overnight before moving to protein purification.

## Protein purification

Mechanical lysis and Nickel-NTA agarose beads purification

For the small-scale induction, we used bead beater as the mechanical lysis method and the proceed to purify the samples with Nickel-NTA agarose beads, due to the high-protein binding and affinity purification of His-tagged fusion proteins. After the lysis process, we will expect to obtain a purified concentration of our protein of interest in the soluble fraction of the purification.

Chemical lysis

We also included trials of chemical lysis using the B-PER Bacterial Protein Extraction Reagent (Thermo Scientific) method when we didn’t obtain a successful separation of our protein of interest into the soluble fraction of the Nickel-NTA purification. We used this process as a way to troubleshoot the mechanical lysis with the bead beaters, as this was not very efficient to release our protein of interest.

Mechanical lysis using French Pressure Cell Press

For the large-scale induction we used a French Pressure Cell Press (Thermo IEC) to have a complete lysis of a big volume culture with the same conditions. Setting the cell pressure between 18.000 to 20.000 as the established pressure used for E. coli.

His-Tag purification Chromatography

For the large-scale induction we used a Ni-NTA affinity purification of a bind-wash-elute procedure in the Fast Protein Liquid Chromatography (FPLC-AKTA chromatography system), using a HisTrap™ FF 5ml columns GE Healthcare) with a sepharose matrix pre-charged with nickel ions. The column was equilibrated in 5 column volumes (CV) Buffer A (Running Buffer for chromatography-See protocols) before the filtered supernant obtained in the mechanical lysis was loaded into the column. The column was then washed with 15 CV of Buffer A before the His-Tagged protein was eluted during a 0-100% gradient of Buffer B (Elution Buffer for chromatography-See protocols) over 15 CV.

Fractions from the chromatography are analysed on an SDS-Page gel and Western blot.

SDS-Page

Is a method used to separate proteins from a complex sample mixture, based on the size of the proteins. All the samples had to be denaturated before loading into the gel and be negatively charged to be separated by size while they move towards the positive probe in the SDS-Page apparatus.

The SDS-Page gels stained with Coomassie stain solution proofs the location of the protein of interest. Otherwise, SDS-Page gels were also used as the first step for Western Blotting.

## Western blot

Is a standard technique used to identify specific proteins by exposing the physical membrane where the proteins bands were transferred to an antibody that will specifically target the protein of interest.

For our experiment, we wanted to confirm the presence of our protein of interest after the chromatography process by targeting the His-tag motif included in our Gblock with a specific antibody targeting it. This would be evidence that the results obtained on the purification process and SDS-Page screening corresponded to our resulting protein of interest.

## Future perspectives

MST assay

The Microscale Thermophoresis Assay (MST) is biophysical assay that quantify the protein-ligand interaction by measuring the motions of molecules in microscopic temperature gradients that fluctuates upon ligand binding. This assay does not require high amounts of purified analyte samples and can be done directly in solution without any additional surface for mobilization. We want to proof the interaction between our obtained and purified GR-LBD protein with the ligand of interest, as a proof of concept that our construct works and can be used in further trials and experiments.

## References

He Y, Yi W, Suino-Powell K, Zhou XE, Tolbert WD, Tang X, Yang J, Yang H, Shi J, Hou L, Jiang H, Melcher K, Xu HE. Structures and mechanism for the design of highly potent glucocorticoids. Cell Res. 2014 Jun;24(6):713-26. doi: 10.1038/cr.2014.52. Epub 2014 Apr 25. PMID: 24763108; PMCID: PMC4042175.

## Protocols

## Resuspend Primers

# Resuspend primers

## Materials

* Sterile distilled water or TE buffer
* GR-Amp-F2 22.4 nmol
* GR-Amp-R2 23.5 nmol
* pET28-Linear-F 23.9 nmol
* pET28-Linear-R2 26.5 nmol
* pET28-seq-R 36.2 nmol

## Procedure

### Primer resuspension for stock

1. Centrifuge the tube containing the primer powder to avoid any volume to not mix properly.
2. For a stock solution of 100µM, the calculations are made following the nmol amount of each primer.

| Primer with correspondent nmol | Stock volume calculation |
| --- | --- |
| GR-Amp-F2 22.4 nmol | 22.4nmol/(100µM) = 224 µL |
| GR-Amp-R2 23.5 nmol | 23.5nmol/(100µM) = 235 µL |
| pET28-Linear-F 23.9 nmol | 23.9nmol/(100µM) = 239 µL |
| pET28-Linear-R2 26.5 nmol | 26.5nmol/(100µM) = 265 µL |
| pET28-seq-R 36.2 nmol | 36.2nmol/(100µM) = 362 µL |
| pET28-seq-F 30.3 nmol | 30.3nmol/(100µM) = 303 µL |

3. The calculated volume for each primer is the necessary volume of ddH2O or TE Buffer to reach a stock primer concentration of 100µM.
4. Add the respective volume into each tube and vortex briefly until the solution is homogeneous.
5. Store the stock primers at -20°C.

### Primer resuspension for working solution

1. Every primer solution was diluted in a 1:10 dilution ratio of the stock solution in new Eppendorf tubes.

V1 = (C2 \* V2) / C1
(10µM \* 10µL) / 0.1mM = 100µL
100µL - 10µL = 90µL

2. Store the working solution in the freezer or fridge for as long as needed.

## Preparation of the Cloning Vector

# Preparation of the cloning vector pET-28a

## Materials

* Qiaprep Spin Miniprep Kit (QIAGEN)
* pET-28a vector culture
* 5x RxN Buffer
* 10mM dNTPs
* 10µM pET28-Linear-F
* 10µM pET28-Linear-R
* Template (1mg/µl)
* Q5 polymerase
* dd water

## Procedure

### Qiaprep Spin Miniprep Kit (QIAGEN)

1. Distribute the 3ml culture of pET28 vector into two separate Eppendorf tubes.
2. Centrifuge into pellet at 6800 x g for 3 minutes at room temperature.
3. Remove supernatant in a safety disposal container, combine and resuspend the pellet in 250µl of Buffer P1.
4. Add 250µl of Buffer P2 and incorporate it by inverting tube 4-6 times.
5. After about 4-5 minutes 350µl of N3 was incorporated it the same way.
6. Centrifuge for 10 minutes at 12.300 x g.
7. Apply all the supernatant to the QIAprep 2.0 spin column by pipetting, and centrifuge for one minute at 12.300 x g.
8. Discard the flowthrough, add 500µl of Buffer PB to the column by pipetting and centrifuge for one minute at 12.300 x g.
9. Discarded the flowthrough, add 750µl of Buffer PE by pipetting and centrifuge for one minute at 12.300 x g.
10. Discard the flowthrough and dried the column by centrifugation in one minute at 12.300 x g.
11. Transfer the column to a collection tube, add 50µl of water to the center of the column and let it stand for one minute before eluting the product by centrifugation in one minute at 12.300 x g.

### PCR

* PCR for up concentration of the cloning vector.

| PCR solution (100 µl) | Required volume |
| --- | --- |
| 5x RxN Buffer | 10 µl |
| 10mM dNTPs | 1 µl |
| 10µM pET28-Linear-F | 2.5 µl |
| 10µM pET28-Linear-R | 2.5 µl |
| Template (1mg/µl) | 0.5 µl |
| Q5 polymerase | 0.5 µl |
| dd water | 83 µl |

* Thermocycler program used for the PCR

#### Thermocycler program (28 cycles)

| Step | Temperature (°C) | Time |
| --- | --- | --- |
| Denaturation | 98 °C | 0:10 |
| Q5 Anneling | 58 °C | 0:30 |
| Extending | 72 °C | 1:40 |
| Final extending | 72 °C | 2:00 |

### Electrophoresis gel

1. Combine 0.4g agarose and 50ml TBE Buffer in an Erlenmeyer flask and distribute the agarose granules by swirling the flask.
2. Promptly put the flask in the microwave and periodically swirl the flask to prevent any regions of higher agarose concentration forming.
3. Once solution is boiling, cool the flask down under flowing water.
4. When tempered, add 5µl of GelRed to the solution.
5. Poured the solution into the previously prepared vessel, and let it set for 40 minutes.

## Gibson Assembly

# Gibson Assembly

## Introduction

Gibson assembly was utilized to clone the Gblock, containing the glucocorticoid gene, into the linearized vector which had been formed from the PCR of the circularized vector. The resulting circularized vector was then to be used further to transform competent TOP10 E. coli cells.

## Materials

* Gblock pET-28-His-GR solution (100 ng/µl) Integrated DNA Technologies (Ref# 239413871)
* Gibson Assembly Master Mix (2X) (NEBio #E2611)
* Vector solution pET-28a(+) (126.79 ng/µl) 9 µl (pET-28a)

## Procedure

1. Take 1 µl of the stock Gblock solution (100 ng/µl) to make a 1x working solution by adding it to a new Eppendorf tube with 99 µl of sterilized distilled water.
2. The quantity of the Gblock needed for the Gibson assembly mix is calculated using the NEBioCalculator: <https://nebiocalculator.neb.com/#!/ligation> with the following input:
   * Insert DNA length: 855 bp
   * Vector DNA length: 5 kb
   * Vector DNA mass: 120 ng (estimate from 126.79 ng/µl provided by NanoDrop)Total volume determined = 1.43µl
3. Gibson assembly mix consists of mixing the following reagents:

| Gibson assembly mix | Required volume |
| --- | --- |
| Gibson Assembly MasterMix (2x) | 2.43µl |
| Gblock pET28-His-GR | 1.43 µl |
| Expression vector pET28a (+) | 1.00 µl |

4. Incubate the Gibson assembly at 50˚C for 1h.

## Transformation of cells

1. Add 4.86µl of Gibson assembly mix to 100µl of Top10 E.coli competent cells.
2. Incubate in ice for 30 minutes.

## Colony PCR

# Colony PCR

## Introduction

We ran the colony PCR to verify if the prepared colonies had successfully incorporated our sequence of interest, our constructed Gblock.

## Materials

* LB Broth medium
* Kanamycin 100µg/ml
* PCR Lysing buffer
* PCR-strip tubes
* Bacteria culture

## Procedure

### Agarose 0.8% gel

1. Combine 0.4g agarose and 50ml TBE Buffer in an Erlenmeyer flask and distribute the agarose granules by swirling the flask.
2. Promptly put the flask in the microwave and periodically swirl the flask to prevent any regions of higher agarose concentration forming.
3. Once solution is boiling, cool the flask down under flowing water.
4. When tempered, add 5µl of GelRed to solution.
5. Pour the solution into the previously prepared vessel, and let it set for 40 minutes.

### LB-Kan liquid culture medium

1. Add 1 ml of Kanamycin 100µg/ml into 1L of LB broth culture medium.
2. Keep refrigerated until further use.

### PCR lysing buffer preparation

1. Follow the instructions for the preparation of the buffer in a suitable container.
2. Constantly stirring while adding the reagents.

| PCR lysing buffer | Required volume |
| --- | --- |
| dd H2O | 1752 ml |
| 10% Triton | 200µl |
| 1M Tris pH 8.0 | 40µl |
| 500 mM EDTA | 8µl |

3. Prepare 7 Eppendorf tubes with 3 ml of LB broth.
4. Also prepare 7 PCR tubes with 30µl of PCR lysis.
5. Transfer 7 independent colonies to the tubes with a loop into the PCR tubes, dipping the colony first into the corresponding LB Broth Eppendorf tube in order to keep a matching culture growing.
6. An 8th colony was picked from the original culture previously processed on miniprep, as a control.

### PCR protocol

1. Made the PCR MasterMix following the presented instructions:

| PCR MasterMix (200 µl x 8 tubes) | Required volume |
| --- | --- |
| Q5 5x Buffer | 40 µl |
| 10mM dNTPs | 4 µl |
| 10µM pET28-Linear-F | 10 µl |
| 10µM pET28-Linear-R | 10 µl |
| Template (1mg/µl) | 4 µl (0.5 per tube) |
| Q5 polymerase | 2 µl |
| dd water | 130 µl |

2. Add 24.4 µl PCR MasterMix into each PCR strip tube first and then add 0.5µl DNA template.
3. Follow the thermocycler program to run the PCR:

| Step | Temperature (°C) | Time |
| --- | --- | --- |
| Initial denaturation | 95 °C | 0:30 |
| Denaturation | 95°C | 0:30 |
| Q5 Anneling | 50 °C | 1:00 |
| Extending | 68 °C | 1:00 |
| Final extending | 68 °C | 5:00 |

4. Add 3µl of 6x Buffer dye to the PCR product and then pipetted 3µl of the mixture to the well in the gel. Repeat the same procedure with all tube samples.
5. Ran the gel for 40 minutes at 100V.

## Gradient PCR

# Gradient PCR

## Introduction

We ran the GradientPCR to verify if the prepared colonies had successfully incorporated our sequence of interest, our constructed Gblock. Also, while assessing the optimal temperature of annealing for our PCR Protocol.

## Materials

* LB Broth medium
* Kanamycin 100µg/ml
* PCR Lysing buffer
* PCR-strip tubes
* Bacteria culture

## Procedure

### Agarose 0.8% gel

1. Combine 0.4g agarose and 50ml TBE Buffer in an Erlenmeyer flask and distribute the agarose granules by swirling the flask.
2. Promptly put the flask in the microwave and periodically swirl the flask to prevent any regions of higher agarose concentration forming.
3. Once solution is boiling, cool the flask down under flowing water.
4. When tempered, add 5µl of GelRed to the solution.
5. Poured the solution into the previously prepared vessel, and let it set for 40 minutes.

### LB-Kan liquid culture medium

1. Add 1 ml of Kanamycin 100µg/ml into 1L of LB broth culture medium.
2. Keep refrigerated until further use.

### PCR lysing buffer preparation

1. Follow the instructions for the preparation of the buffer in a suitable container.
2. Constantly stirring while adding the reagents.

| PCR lysing buffer | Required volume |
| --- | --- |
| dd H20 | 1752 ml |
| 10% Triton | 200µl |
| 1M Tris pH 8.0 | 40µl |
| 500 mM EDTA | 8µl |

1. Prepare 7 Eppendorf tubes with 3 ml of LB broth.
2. Also prepare 7 PCR tubes with 30µl of PCR lysis.
3. Transfer 7 independent colonies to the tubes with a loop into the PCR tubes, dipping the colony first into the corresponding LB Broth Eppendorf tube in order to keep a matching culture growing.
4. An 8th colony was picked from the original culture previously processed on miniprep, as a control.

### PCR protocol

1. Made the PCR MasterMix following the presented instructions.

| PCR MasterMix (200 µl x 8 tubes) | Required volume |
| --- | --- |
| Q5 5x Buffer | 40 µl |
| 10mM dNTPs | 4 µl |
| 10µM pET28-Linear-F | 10 µl |
| 10µM pET28-Linear-R | 10 µl |
| Template (1mg/µl) | 4 µl (0.5 per tube) |
| Q5 polymerase | 2 µl |
| dd water | 130 µl |

1. Add 24.4 µl PCR MasterMix into each PCR strip tube first and then add 0.5µl DNA template.
2. Follow the thermocycler program to run the PCR.

### Thermocycler program (30 cycles)

| Step | Temperature (°C) | Time |
| --- | --- | --- |
| Initial denaturation | 95 °C | 0:30 |
| Denaturation | 95°C | 0:30 |
| Q5 Anneling | 50 °C | 1:00 |
| Extending | 68 °C | 1:00 |
| Final extending | 68 °C | 5:00 |

1. Add 3µl of 6x Buffer dye to the PCR product and then pipetted 3µl of the mixture to the well in the gel. Repeat the same procedure with all tube samples.
2. Ran the gel for 40 minutes at 100V.

## Enzyme Digestion

# Enzyme Digestion

## Materials

* Linear vector circular plasmid
* Hind III
* Control circular plasmid uncut
* PCR-strip tubes
* Bacteria culture

## Procedure

### Initial verification of backbone plasmid

1. We ran electrophoresis using linear vector sample circular plasmid + Hind III and control circular plasmid uncut.

| Sample Group | Composition |
| --- | --- |
| Sample of linear vector | 1.5 µl from concentration of 126 µg/µl linear vector + 8.5 µl of water |
| Sample of cut circular plasmid | 4 µl from 40 µg/µl circular plasmid + 4 µl of water + 1 µl CutSmart Buffer + 1 µl 20 k/µl Hind III |
| Sample of control circular plasmid | 4 µl C. plasmid 40 µg/µl + 5 µl water + 1 µl CutSmart Buffer |

### Enzyme Digestion

1. Incubate the cut and control C-plasmid samples in a PCR machine with a heat block temp 37°C and lid temperature of 38°C for 15 minutes.
2. After incubation, add 2 µl of GelRed dye to each sample: Linear vector, cut C. Plasmid, and control C. plasmid.
3. Take 6 µl of each and add to 0.8% agarose gel with 1.5 µl 1kb ladder.
4. Follow Culture PCR protocol to run PCR process for the seven independent colonies from our cryostock of pET27-GR.
5. Inoculate the 7 colonies from our cryostock.
6. After electrophoresis, all 7 colonies showed a strong band at ~1000 kb, indicating that all 7 colonies had incorporated the vector.

## Transformation of BL21(DE3)

# Transformation of BL21(DE3)

## Materials

* BL21(DE3) Competent E. coli
* 2 PCR colony plasmid products (33ng/ µl)
* LB-Kanamycin Agar plate
* SOC medium
* 50% glycerol

## Procedure

### Transformation of BL21(DE3) competent E.coli

1. Thawing of two tubes of PCR colony plasmids.
2. Thawing of BL21(DE3) Competent E. coli tube.
3. After completely thawed, keep both tubes on ice.
4. Add 100 µl of BL21 in a new Eppendorf tube, and then add 1 µl of plasmid.
5. Incubate on ice for 30 minutes.
6. Heat shock at 42°C for 90 secs in water bath.
7. Transfer tube to ice for 2 minutes.
8. In a clean security bench, add 900 µl of SOC to the tube.
9. Incubate at 37°C 880 RPM for 1 hour.
10. Plate 100 µl on a pre-warmed kanamycin plate at 37°C.
11. Incubate at 37°C overnight.
12. Transfer to fridge the next morning.

### Pre-culture for stock

1. Get the BL21DE3 + pET28-GR plate and the LB-Kan culture media from the fridge, along with a 13 ml culture tube.
2. Pipette 5 ml of LB-Kan media into the culture tube, in a clean security bench.
3. Inoculate culture tube with a single colony from the plate using a loop.
4. Place culture tube in the shaking incubator at 37°C, 200 rpm overnight.
5. Place LB-Kan bottle and plate back in the fridge.

### Cryo-Stock of protein expression strain BL21DE3-pET28-GR

1. Add 500 µl of the overnight culture solution into a 2 ml Screw cap tube.
2. Add 500 µl of 50% glycerol and mix well by inversion.
3. Label the tube clearly and place it at -80°C.

## Culture Preparation and Induction

# Culture Preparation and Induction

## Materials

* Culture plate with BL21DE3-pET28-GR colonies
* LB broth kanamycin-50 liquid medium
* 1 M IPTG
* 2.88 mM prednisolone
* Centrifuge Avanti J-26S XP, Beckman Coulter
* 1x PBS

## Procedure

### Pre-culture Liquid Solution

* Before starting, have a freshly streaked culture plate of BL21DE3-pET28-GR colonies. If possible, prepare the plate a day before the culture preparation and leave it incubating at 37°C overnight.
* Pipette 15 ml of LB-Kan into a 100 mL flask in the security cabinet bench.
* Inoculate the culture flask with a single colony from the plate using a loop.
* Place culture tube in shaking incubator at 37°C and 200 rpm overnight.

### Dilution of the Culture and Induction

* 1:100 dilution by taking 15 ml of the overnight pre-culture and adding it to 1L of LB-Kan in a suitable container. Shake softly with each addition.
* Separate the inoculated solution into 500 mL portions into two 2L conical flasks to fit the incubator and to allow optimal bacterial growth.
* Incubate the cultures at 37°C 220 rpm until reaching the optimal Optical Density (OD)

+ *Optimal OD score = 0.4 – 0.7*
+ *Considering the pre-culture volume suggested, it is expected that the culture will reach the optimal OD score after 2 hours and 2 hours 50 minutes. Check constantly during the period to stay within the optimal range.*

* Once the culture has reached an optimal OD, add 12.5 μl of IPTG and 177 μl of prednisolone to each flask. Also, add a drop of an anti-foaming agent.
* Incubate the flask at 20°C overnight.

### Centrifugation and Storage

* After the overnight incubation, transfer the liquid cultures to a 1-liter container suitable for use in the Centrifuge Avanti J-26S XP.
* Load the container and balance the centrifuge with a container filled with water until reaching the approximate weight of the container with the liquid culture and set the following conditions on the centrifuge.

| Rotor ID | Speed | Time | Temperature |
| --- | --- | --- | --- |
| JLA 8.1000 | 5,000 x g | 15 minutes | 4°C |

* Discard the supernatant into containers with bleach for safe disposal.
* Add 35 mL of PBS into the pellet to dissolve gently. Use the same pipette or vortex the container to resuspend and mix the PBS constantly.
* Transfer the resuspended pellet to a 50 mL Falcon tube.
* Centrifuge at top speed for 15 minutes.
* Discard the supernatant and store the pellet in a freezer at -20°C.

## SDS-Page Gel for Coomassie

# SDS-Page Gel for Coomassie Blue Staining

## Materials

* SDS-Page apparatus
* 1x SDS-Page running buffer
* 4x SDS-Page loading buffer
* Coomassie staining solution
* Novex 4-20%, Tris-Glycine PlusWedgeWellTM Gel, Invitrogen (Ref XP04202BOX)
* PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (Catalog Num=26619)

## Procedure

### Sample Preparation

* Sample of interest is aliquoted in 20 μl volumes in new working Eppendorf tubes to have a precise calculation of the amount of loading buffer and samples that will be loaded into the gel.
* Add 5 μl of 4x SDS-Page loading buffer to each working tube.
* Incubate samples at 75°C for 5 minutes.

### Gel Loading

* Prepare SDS-Page apparatus and load the Novex 4-20%, Tris-Glycine PlusWedgeWellTM Gel into the apparatus.
* Fill the SDS-Page apparatus with 1x Running buffer until the gel and the bottom of the apparatus is completely covered.
* Load the first well with 3 μl of the Prestained Protein ladder.
* Load 5 μl of the working samples to fill up the rest of the wells.
* Run at 225 V for approximately 40-45 minutes.

### Coomassie Stain

* Put the gel on a separate clean container or recipient, wash shortly with a bit of distilled water and then discard the liquid.
* Cover the gel with the Coomassie staining solution (approximately 25 ml) and put the container in a moving rack for 10 minutes.
* Pour the staining solution off into a chemical container, wash the gel with distilled water and then discard it.
* Cover the gel with destaining solution and put the container in a moving rack for 1 hour.
* Pour the destaining solution off into a chemical container, wash the gel with distilled water and then keep it covered until visualization.
* The gel can be kept covered with distilled water overnight for better resolution during the imaging process.

## Protein Purification

# Protein Purification

## Materials

* Lysis buffer
* Running buffer
* Elution buffer
* DNAse
* French Pressure Cell Press, Thermo EIC
* Centrifuge Avanti J-26S XP, Beckman Coulter
* Puradisc syringe filters of 0.45 μm and 0.20 μm
* HisTrapTM FF 5ml column, GE Healthcare
* FPLC-AKTATM chromatography system, GE Healthcare

## Procedure

### Sample Preparation

* Add samples of bacterial culture into a 50 ml falcon tube. Record the empty and filled weights of the tube to have an approximation of the culture weight for the next steps.
* Centrifuge at top speed for 15 minutes.
* Discard the supernatant completely and weigh the approximate weight of the pellet.
* Add 30 mL of lysis buffer and 50 μl of DNAse per mL of lysis buffer (1500 μl) to the resulting pellet. The final volume of the working sample must be a maximum of 35 mL.
* Put the falcon tube on the roller mixer for half an hour.

### Mechanical Lysis

* Set the piston in the security location and add glycerol to the moving sections for better movement.
* Start the lysis by loading the sample into the piston by changing the ratio selector to allow a pressure difference. Set the cell pressure between 18,000 to 20,000 as the established pressure used for E. coli, Piston high ratio between 1150 to 1280.
* Repeat the lysis three times. Be aware of the pressure levels at any step of the process.
* Release the pressure completely at the end of the run.
* Take 1 mL of the resulting lysed solution in a separate Eppendorf tube. This sample will be used as a reference of an intermediate stage and to check the presence of the protein of interest at stages of the purification process with an SDS-Page gel. Label accordingly to keep track of the stage it represents.

### Centrifuge Protocol

* Load the resulting lysed sample into the centrifuge and set the following conditions for the centrifuge process:

| Rotor ID | Speed | Time | Temperature |
| --- | --- | --- | --- |
| JA 25-50 | 30,000 x g | 30 minutes | 4°C |

* Take 1 mL of the resulting solution after the centrifugation in a separate Eppendorf tube. This sample will be used as a reference of the intermediate stage and to check the presence of the protein of interest at stages of the purification process with an SDS-Page gel.
* Take a section of the pellet with a loop and dilute it in 1 mL of PBS. This sample will be used as a reference of the intermediate stage and to check the presence of the protein of interest at stages of the purification process with an SDS-Page gel.
* The resulting supernatant is filtered twice using the 0.45 μm and 0.20 μm syringe filters, respectively.
* Attach a plastic syringe to the filters and pour the supernatant to pass across the filter into a new sterilized falcon tube.
* Take 1 mL of the resulting solution from the filtrate process. This sample will be used as a reference of the intermediate stage and to check the presence of the protein of interest at stages of the purification process with an SDS-Page gel.

### His-Tag purification Chromatography

* Equilibrate the HisTrapTM FF 5ml column with 5 column volumes (CVs) or 25 mL of the running buffer.
* Load the filtered supernatant onto the column.
* Wash the column with 15 CVs of the running buffer.
* Elute the protein by applying a 0-100% gradient of the elution buffer over 15 CVs.
* Acquired fractions are to be analyzed using SDS-PAGE and Western blot.

## Western Blot

# Western Blot

## Materials

* Blotting Buffer
* Blocking Buffer
* Transfer Buffer
* TBST
* His-Tag Horseradish Peroxidase-conjugated Antibody, Biotechne (Product # MAB050H)
* PVDF Transfer Membrane, 0.45 μM, for Western Blotting applications. Thermo Scientific (Product #88518)
* Novex 4-20%, Tris-Glycine PlusWedgeWellTM Gel, Invitrogen (Ref XP04202BOX)
* ECL TM Prime Western Blotting Detection Reagents, Cytiva (Ref RPN2232)
* PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa (Catalog Num=26619)
* Methanol
* Filter paper

## Procedure

### SDS-Page

1. Load protein into the well of the SDS-Page gel, along with the PageRuler™ Protein Ladder.
2. Run the gel at 190 V until the bands reach the end of the gel (approximately 40-50 minutes).

### Western Blotting

1. Cut the membrane and the filter paper sheets to the approximate size of the gel.
2. Activate the membrane with methanol for 1 minute and rinse it afterwards with transfer buffer before preparing the stack.
3. Stack the membrane in the following order:
   * Three sections of Filter paper - PVDF - SDS-Page Gel - Three sections Filter Paper
4. Wet all the sections of the stack with transfer buffer after assembly.
5. Place the stack in the TE 70x semi-dry transfer unit from Hoefer and start electrophoretic transfer with a voltage of 30 V for 1 hour.
6. Block the membrane for 1 hour at room temperature or overnight at 4°C.
7. Incubate the membrane with the His-Tag Horseradish Peroxidase-conjugated Antibody for 1 hour at room temperature or overnight at 4°C.
8. Wash the membrane with TBST 3 times, 5 minutes each time in a shaker.
9. Remove the membrane with WB forceps, slightly absorb excess liquid with absorbent paper, and place the membrane on clean cling film.
10. Depending on the size of the film, add 1 ml of ECL-prime working solution per 10 cm2 of the film in drops onto the membrane, ensuring that the solution covers the membrane evenly, and leave it for 5 minutes at room temperature.

Working ECL-prime = 1.5 ml Solution A (luminol) + 1.5 ml Solution B (peroxide)

11. Remove the excess liquid with absorbent paper. Place the membrane between two pieces of cling film, then place the membrane into the chemiluminescence imager, and take pictures.

## Buffer Recipes

## 1x Transfer Buffer for Western Blotting

# 1x Transfer buffer for western blotting

## Introduction

Solution is used to transfer the separated proteins from the SDS-Page gel to a PVDF membrane to obtain a good visualization of the proteins of interest.

## Reagents

|  |  |  |
| --- | --- | --- |
| 1 L | Transfer buffer | Required volumes/weight |
| 48 mM | Tris base | 5.76 g/L |
| 39 mM | Glycine | 2.95 g |
| 20% | Ethanol | 200 ml |

## Procedure

* Add the Tris base into the 200 ml ethanol with distilled water.
* Add the glycine and complete the desired volume with distilled water.
* Keep refrigerated at approximately 4°C

## 4x SDS-PAGE Loading Buffer

# 4x SDS-Page loading buffer

## Introduction

Used for preparing and loading protein samples into polyacrylamide gels for visualization and posterior analysis.

## Reagents

|  |  |  |
| --- | --- | --- |
| 15 ml | 4X SDS-Page loading buffer | Required volumes/weight |
| 200 mM | Tris-Cl (pH 6.8) | 3 ml |
| 400 mM | DTT | 925 mg |
| 8% | SDS | 1.2 g |
| 0.4% | Bromophenol blue | ~60 mg |
| 40% | Glycerol | 6 ml |

## Procedure

* Add the reagents into 15 ml Falcon tube.
* The required volume of Bromophenol blue is an approximation, as it was added while checking the color of the buffer until it was sufficiently blue.
* Keep at room temperature.

## 10x SDS-Page Loading Buffer

# 10x SDS-Page Running Buffer

## Introduction

Used as the stock solution to prepare 1x Running Buffer, the electrophoresis buffer for the SDS-Page visualization procedure.

## Reagents

|  |  |  |
| --- | --- | --- |
| 1L | 10x SDS-Page Running Buffer | Required volumes/weight |
| 0.2051 M | Tris base | 30.3 g/L |
| 1.924 M | Glycine | 144.4 g/L |
| 0.03467 M | SDS | 10 g/L |

## Procedure

* Add 800 mL of distilled water in a 1L container
* Add the Tris base to the solution while stirring. Then add the calculated volume of glycine and SDS next.
* Keep stirring the solution for approximately an hour.
* Complete 1 liter of volume by adding distilled water.
* Keep at room temperature.

## Coomassie Staining

# Coomassie staining solution

## Introduction

Solution used for visualization of proteins in an SDS-Page gel and protein detection.

## Reagents

|  |  |  |
| --- | --- | --- |
| 1L | Coomassie Stain | Required volumes/weight |
| 0.125% | Coomassie blue R250 | 1.25 g/L |
| 10% | Acetic acid | 100 mL/L |
| 40% | Ethanol | 500 mL/L |

## Procedure

* Dissolve the Coomassie blue R250 in 500mL ethanol while stirring.
* Stir the solution for 3-4 hours until it has been homogenized. Filter the solution through Whatman filter paper to remove any insoluble material
* Add the acetic acid
* Complete the volume with distilled water until reaching the total volume
* Keep at room temperature.

## Destaining Solution

# Coomassie Destaining solution

## Introduction

Destaining agent important to remove the additional dye present in the gel and to obtain a clear background on the imaging of the SDS-Page gel

## Reagents

|  |  |  |
| --- | --- | --- |
| 1L | Coomassie Destain | Required volumes/weight |
| 10% | Acetic acid | 100 mL/L |
| 40% | Ethanol | 500 mL/L |

## Procedure

* Add 500 mL of ethanol in 400 mL of distilled water
* Add 100 ml of acetic acid to reach the total volume required.
* Keep at room temperature.

## Elution Buffer for Protein Purification and Chromatography

# Elution buffer for protein purification and chromatography

## Introduction

One of the crucial buffers for affinity purification of proteins.

## Reagents

|  |  |  |
| --- | --- | --- |
| 1L | Elution Buffer | Required volumes/weight |
| 20 mM | NaPi pH 8.0 | 2.84 g/L |
| 150 mM | NaCl | 8.77 g/L |
| 500 mM | Imidazole | 34.04 g/Ltd> |
| 10% | Glycerol | 100 ml/L |
| 1 mM | DTT | 154.25 mg/L |
| 10 μM | Ligand | 3.571 mL of 2.8 mM stock/L |

## Procedure

* Add the powder reagents and the glycerol into a suitable container to be stirred
* Add 800 mL of distilled water while stirring until the solution gets homogenize
* Add the ligand and DTT last to avoid changing the temperature of the tubes while adding the rest of the reagents
* Complete the total desired volume with distilled water
* The resulting solution must be filtered and degassed before putting it in a sterilized suitable container to avoid adding bubbles into the chromatographer.
* Keep refrigerated at approximately 4°C

## Lysis Buffer

# Lysis Buffer

## Introduction

Buffer used for protein purification protocols

## Reagents

|  |  |  |
| --- | --- | --- |
| 50 ml | Lysis buffer | Required volumes/weight |
| 50 µM | Prednisone | 893 µl |
| 10% | Glycerol | 100 mL (99.5% glycerol) |
| 50 mM | Tris | 2.5mL of 1M Tris |
| 400 mM | NaCl | 4 mL of NaCl (3M) |
| 0.5% | Chaps | 250 mg |

## Procedure

* Add the corresponding reagents on a 50 mL falcon tube.
* Put the falcon tube on a tube roller mixer for approximately 30 minutes, until the buffer looks completely homogeneous.
* Filter the buffer before pouring into a sterilized 50 mL falcon tube.
* Kept on cold room at 4°C approximately

## Milk for Blocking Western Blots

# Blocking buffer for Western blotting

## Introduction

Blocking buffer is necessary to prevent non-specific binding of antibodies to the membranes and allow a better visualization of the proteins of interest.

## Reagents

|  |  |  |
| --- | --- | --- |
| 50 mL | Blocking buffer | Required volumes/weight |
| 5% | Milk for Blocking Western Blots | 2.5 g/50ml |
| 1X | TBST | 50 mL |

## Procedure

* Add the powder reagent to a 50 ml falcon tube before adding the total volume of TBST.
* Put the falcon on a tumbler for approximately half an hour or until the buffer is completely homogeneous.
* Keep refrigerated at approximately 4°C

## Running Buffer for Chromatography

# Running buffer for chromatography

## Introduction

One of the crucial buffers for the separation of proteins during the chromatography process

## Reagents

|  |  |  |
| --- | --- | --- |
| 1L | Running buffer | Required volumes/weight |
| 20 mM | NaPi pH 8.0 | 2.84 g/L |
| 500 mM | NaCl | 29.22 g/L |
| 50 mM | Imidazole | 3.4 g/L |
| 10% | Glycerol | 100 ml/L |
| 1 mM | DTT | 154.25 mg/L |
| 10 μM | Ligand | 3.571 mL of 2.8 mM stock/L |

## Procedure

* Add the powder reagents and the glycerol into a suitable container to be stirred
* Add 800 mL of distilled water while stirring until the solution gets homogenize
* Add the ligand and DTT last to avoid changing the temperature of the tubes while adding the rest of the reagents
* Complete the total desired volume with distilled water
* The resulting solution must be filtered and degassed before putting it in a sterilized suitable container to avoid adding bubbles into the chromatographer.
* Keep refrigerated at approximately 4°C

## TBST for Western Blotting

# TBST for Western blotting

## Introduction

Wash buffer for the PVDF membrane after the incubation process

## Reagents

|  |  |  |
| --- | --- | --- |
| 500 mL | TBST | Required volumes/weight |
| 25 mM | Tris Base | 1.51 g/50mL |
| 150 mM | NaCl | 4.38 g/50ml |
| 0.1% | Tween20 detergent | 0.5ml/50mL |

## Procedure

* Add the reagents to an initial volume of 250 ml of distilled water to container suitable for stirring.
* Complete desired volume with distilled water.
* Keep refrigerated at approximately 4°C

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# Results

## 1. Cloning and transformation

### Aim

* Insert our gBlock pET-28-His-GR into pET-28a (+) cloning vector
* Transform the cloning strain TOP10 E. coli with our inserted plasmid
* Transform successfully the expression strain BL21(DE3) and identify the desired plasmid

### Conclusion

* We successfully inserted our gBlock coding for the Glucocorticoid Receptor Ligand Binding (GR-LBD) with an additional His-Tag using Gibson Assembly Method.
* TOP10 E. coli cloning strain was successfully transformed with our construct plasmid containing our sequence of interest.

We isolated our cloning vector on interest, pET-28a (+) from stock cultures of TOP10 E. coli available in the lab using Qiaprep Miniprep, up-concentrated the resulting linearized vector and used 0.8% agarose gel electrophoresis to confirm the high concentration of the vector in our product.

After confirming the up concentration of our cloning vector of interest, we proceed to clean the PC product with MinElute column and then proceed to assemble our gBlock with the cloning vector through Gibson assembly. TOP10 E. coli cells were transformed with our resulting construct, and single-colony PCR with primers targeting insertion site was performed at the same time as colonies were inoculated in liquid culture.

Insertion of gBlock sequences were successfully determined in gel electrophoresis with the seven bands at ~1000 kb, corresponding to the seven independent colonies of TOP10-pET-28-His-GR. The results of the gel were also confirmed by sanger sequencing of the isolated plasmids.

![08%agarose](https://static.igem.wiki/teams/5333/lab-results/08-agarose.webp)

**Figure 1:** 0.8% agarose gel showing the band obtained from gel electrophoresis after running for 100 V for 40 minutes. Lane 1 contains the GeneRuler 1 Kb ladder. Lane 2 contains the PCR product of linearization of cloning vector Pet-28a (+).

After this, we proceed with the transformation via Heatshock of BL21(DE3) E. coli cells with our construct TOP10- pET-28-His-GR. Due to time limitations we proceed to start Pre-culture experiments with the transformed cells to confirm the transformation of this cells via induction of the culture using IPTG as the inducer and prednisone as the ligand.

Glycerol stocks of both transformation process were made and kept at -80°C for future experiments, particularly the cryo-stock BL21DE3-pET28-GR.

![08%agarose2](https://static.igem.wiki/teams/5333/lab-results/08-agarose-2.webp)

**Figure 2:** 0.8% agarose gel showing bands of products from Colony PCR from seven individual colonies (Lane 2 to 8) of transformed TOP10 E. coli colonies with our pET-28-His-GR gBlock. The final lane shows the negative control.

# 2. Protein expression

### Aim

* Confirm successful transformation of our BL21DE3-pET28-GR construct in E. coli BL21(DE3) expression strain.
* Caracterize the small-scale induction of the cultures to obtain the most optimal temperature and time conditions for the incubation of a successfully induced culture to mass-produce our plasmid of interest correctly in a large-scale culture induction.

### Conclusion

* We opted to standardize the incubations conditions in 20°C overnight since we obtained more concentrated proteins.
* We confirmed the issues with solubility of our protein of interest reported in the literature while replicating the incubation conditions proposed.

After the transformation of BL21(DE3) E. coli cells we proceed with the small-scale induction of expression to optimize the incubation conditions for our cells. We noticed that most of our protein ended up in the insoluble fraction, as shown in Figure 3. Our protein of interest shows clear bands on the insoluble fraction at ~25KDa with a relatively higher concentration on the condition 20°C overnight conditions.

![RackA-25](https://static.igem.wiki/teams/5333/lab-results/racka-25-07-2024.webp)

**Figure 4:** 4-20% SDS-Page gel image of BL21DE3-pET28-GR expression. The numbers correspond to the specific incubation conditions in Flowthrough fraction (1-5) and insoluble fraction (6-10) following the same incubation periods in each fraction: Uninduced 37°C, 20°C/4hours, 20°C/Overnight, 37°C/4 hours, 37°C/Overnight.

# 3. Protein purification and future perspectives

### Aim

* Test if the large-scale induction of our construct allowed for a most efficient expression of our protein of interest.
* Start the mass-production of our protein by large-scale inducing cultures to be purified in Fast Protein Liquid Chromatography (FPLC).
* Confirm that the protein obtained in the elution fractions of the chromatography corresponds successfully to our protein of interest by performing Western blotting on the regular elution fractions and up-concentrated elution fractions.

### Conclusion

* The incubation conditions determined to be optimal in the previous experiments in the small-scale expression showed a successful cultural growth and induction in large-scale expression.
* Successfully confirmed that the protein obtained in the elution fractions obtained in the FPLC corresponds to our protein of interest by performing Western blotting and SDS-Page electrophoresis.

After the transformation of BL21(DE3) E. coli cells we proceed with the small-scale induction of expression to optimize the incubation conditions for our cells. We noticed that most of our protein ended up in the insoluble fraction, as shown in Figure 3. Our protein of interest shows clear bands on the insoluble fraction at ~25KDa with a relatively higher concentration on the condition 20°C overnight conditions.

![SDS-PAGE](https://static.igem.wiki/teams/5333/lab-results/11-sept-2024-sdspage.webp)

**Figure 5:** 4-20% SDS-Page gel image of BL21DE3-pET28-GR protein expression after Nickel-NTA agarose beads with three specific fractions: Flowthrough, First Elution and second Elution.

After the transformation of BL21(DE3) E. coli cells we proceed with the small-scale induction of expression to optimize the incubation conditions for our cells. We noticed that most of our protein ended up in the insoluble fraction, as shown in Figure 3. Our protein of interest shows clear bands on the insoluble fraction at ~25KDa with a relatively higher concentration on the condition 20°C overnight conditions.

We then proceed to start a new cultures while carefully controlling the OD score to obtain a successful colony before adding the inducer and ligand on large-scale cultures. After 2 hours and 50 minutes and an OD score of 0.4235, we proceed to add the inducer and ligand and let it grow under the optimized conditions, 20°C overnight. Mechanical lysis was performed using the French-Press mechanical lysis protocol (Under Protein purification protocol). The filtered supernatant obtained was purified using a HisTag FF column.

The chromatography results of the His-tag purifications shows a specific peak on the elution fraction between T13 to T15 (Figure 6) that likely corresponds to the Eluted protein of interest.

![Chromatography](https://static.igem.wiki/teams/5333/lab-results/24-09-2024-chromatography.webp)

**Figure 6:** Chromatography of BL21DE3-pET28-GR purification with a HisTrap FF 5 ml column. Conducted with ÄKTA start protein purification system.

We confirmed the results of the chromatography on an SDS-Page Coomassie-stained gel with the main sections of the purification process (Flowthrough, washing step and elution fraction). We confirmed that our protein of interest is being expressed and successfully purified by the bands at ~25kDa molecular weight.

![SDS-PAGE-2](https://static.igem.wiki/teams/5333/lab-results/24-09-2024-sdspage.webp)

**Figure 7:** 20% SDS-Page gel image of GR-LBD expression after chromatography purification. The numbers correspond to the Tubes used from the fractions obtained in the chromatography process: 1. Flowthrough (Tube 3), 2. Washing step (Tube 8), 3. Elution fractions (Tube 13 to Tube 20). An additional washing step (4. Tube 10) was added in the final well to improve the visualization of the gel.

At the same time, we started the Western Blot process using the same samples form the purification fractions in an SDS-Page run at 190 v. We incubated the transfer membrane with the MAB050H His-Tag Horseradish Peroxidase-conjugated antibody to target our protein of interest in the fraction parts. As shown in figure 8, it was confirmed that our protein of interest is present in the fractions, however the higher signal detected by the BioRad imager was principally detected in the Flowthrough fractions, with no evident signal in the other fractions.

![SDS-PAGE-2](https://static.igem.wiki/teams/5333/lab-results/24-september24-westernblot.webp)

**Figure 8:** Western blot visualization of the purified GR-LBD fractions obtained by chromatography.

Even though the western blotting might suggest that we don’t have any concentration of the purified protein in the elution fractions, the SDS-Page gel suggest differently. Therefore, we proceed to up-concentrate the eluted protein Tubes 13 to by precipitations and filtration once again in each separated samples. We compared the concentration obtained with the regular elution fractions by including the original elution samples on the subsequent SDS-Page gels, one for Coomassie staining and the other for western blotting.

![SDS-PAGE-3](https://static.igem.wiki/teams/5333/lab-results/26-09-2024-sds-page.webp)

**Figure 9:** SDS-Page Coomassie-stained gel showing the differences in the signal between the up-concentrated Elution fractions and the regular elution fractions of the purified GR-LBD (Tube 13 to Tube 16, for both).

Therefore, we can conclude that we have successfully expressed our protein of interest and demonstrated that the expression and purification experiments that we have done so far with the large-scale induction and FPLC purification. We’re still in our way to improve the purification process and the solubility of our protein of interest, but the results that we have obtained so far are encouraging and a good signal that we’re in the correct path to complete our main goal.

![western-blot-2](https://static.igem.wiki/teams/5333/lab-results/26-09-2024-westernblot.webp)

**Figure 10:** SDS-Page Coomassie-stained gel showing the differences in the signal between the up-concentrated Elution fractions and the regular elution fractions of the purified GR-LBD (Tube 13 to Tube 16, for both).

{% endblock %}