iGEM 2024 — Team ehsbnu

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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

## Date: 1.21 First meeting

Introduce everything about this competition and provide specific information.

Assigned tasks: Started researching for our project topic.

## Date: 2.7 Online meeting

Discussed the issue with lab resources. Introduced some lab safety precautions, and assigned some reading/learning tasks to get a general understanding of synthetic biological experiment.

Reading & Learning: Cambell Biology, finish Chapter 3 before the next meeting.

## Date: 2.24 Online meeting

Discussed through a series of methods and strategies used in finding laboratory resources, reading papers, and writing literature reviews, etc.

## Date: 3.7 Meeting

Discussed about the protein transcription process. Discussed on strategies in finding and reading the articles.

Assigned task:
Each team member in the biology department: finish three literature review per person before next Wednesday.

## Date: 3.14, 3.21, 3.28

## Offline meeting: project brainstorm and discussion.

Presented and discussed through the literature reviews.

Presenter: Lisiqi Ye, Yushan Mei, Zhaoyuan Wu, Gaocheng Zeng, Jiayi Chen, Youzi Wang, Zihe Chen, Yiming Wu, Jingyuan Ma, Yujia Chen, Qingshu Wu.

Organized and collected all accessible topics, assigned reading materials to everyone for further investigation into these topics.

Divide the students into groups, each group focused on one research topic.

Group Hosts: Xirui Wang, Fangming Liu, Yiming Wu, Boyue Deng and Junru Huang, Xinyi Zhao, Yuxuan Wu.

Finally agreed on the topic of limulus blood Factor G proposed by Boyue Deng and Junru Huang.

## Date 5.21

Finish the selection about the topic and start designing the experiment: Identify the topic & read some relevant literature

## Date 6.10 Online Meeting

Confirm the website construction person in charge of Lisiqi Ye and Zihe Chen
Decide not to add new team members

Vote to confirm the approval rate of the leaders of Jiayi Chen and Gaocheng Zeng, and confirm the formal transfer of leadership positions to the Senior One

A meeting will be held within two weeks to design the specific content of the experiment

At present, laboratory resources are limited to the school. Urge all students to find laboratory resources

## Date 6.21 Offline Meeting

Build the team wiki.

Design the specific experimental steps.

Complete the project description.

## Date 7.9 Offline Meeting

Search for Tachypleus tridentatus clotting Factor G alpha and beta subunit's gene sequence. Perform BLAST and codon optimization. Start designing the PCR primers.

## Date 7.12

Design cultural products.

## Date 7.25 Offline Meeting:

complete the PCR primers design

## Day 9.3 Start the Experiment

Centrifuge the cell pellets: 13,000 rpm for 5 minutes.

Prepare LB medium (100 mL):

1g NaCl

1g tryptone

0.5g yeast extract

1.2-1.5g agar

100 mL water

Sterilization: Autoclave at 121°C for 20 minutes.

Resuspension of cell pellets: Dilute in ddH₂O (1:1000).

Concentration: 100 ng/μL.

Plasmid transfer: 100 μL of competent BL21 (DE3) cells: Add 2 μL of antibiotic, incubate on ice for 15 minutes.

Heat shock: 42°C for 90 seconds.

Recovery: 1 mL of non-antibiotic LB medium, incubate at 37°C for 1 hour.

Centrifuge and plate the cells:

Centrifuge at 4000 rpm for 5 minutes.

Retain 100 μL of supernatant and discard the rest.

## Date 9.4

Selected six colonies from the agar plates for both proteins (12 test tubes in total).

Inoculated with 5 mL of LB liquid medium and incubated at 37°C.

Measured OD at 595 nm using a spectrophotometer.

OD is within the appropriate induction range, induced with IPTG at a final concentration of 1 mM.5. Prepared a 1 M stock solution of IPTG, diluted with dd H2O (Before induction, took 400 μL of the culture for pre-induction samples).

Filter-sterilized using a 0.22 μm filter and aliquoted.

## Day 9.9

Conducted the SDS-PAGE, the target protein has not been successfully produce

Performed Western Blot for further confirmation:

1. Preparation of transfer buffer: 1 L of transfer buffer consists of 3.55 g of TRIS, 14.4 g of glycine, and 200 mL of methanol, adjusted to a final volume of 1 L with distilled water.

2. Prepare 1 L of TBS (Tris-Buffered Saline) buffer: 2.42g Tris (20mM), 8.76g NaCl (150mM), measured pH=7.45 add 1‰ TWEEN-20 before using.

3. "The Sandwich Structure": Materials needed for Western blot:

A clamp (black cathode at the bottom)

One piece of sponge, pre-soaked in transfer buffer

Three pieces of filter paper, pre-soaked in transfer buffer

The gel

PVDF membrane (requires activation with methanol due to its hydrophobic nature)

Three pieces of filter paper, pre-soaked in transfer buffer

One piece of sponge, pre-soaked in transfer buffer

A clamp (white anode at the top)

Place on ice under a constant current of 300 mA for 2 hours.

4. Blocking: prepare a blocking solution containing 5% (w/v) non-fat dry milk in TBST, seal it and leave it overnight.

## Date 9.15

experimental procedureuse on the competent cells:

Add 2 micrograms of plasmid to each of the 3 competent cells and let them stand for 15 minutes

Place the cells at 42 degrees Celsius for 90 seconds

Add 1ml of culture medium and incubate at 37 ° C for 45 minutes

Centrifuge at 4000 rpm for 3 minutes

Coating board

## Date 9.22

After the last time the agarose gel electrophoresis did not run out, we replaced the materials by the competent cells. And we did all the process again:

heat shock, resistance screening, OD measurement and IPTG induction expression, SDS-PAGE gel run and harvest.

But this time there were some results, the alpha subunit was sure to express and
doing the mass spectrometry on beta submit.

## Date 9.29

beta subunit expressed and make preparation before induction,

measure OD and add inducer.

OD is measured as 7.03.

## Date 10.2

dilute the material ten times

coating board

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Image 1: Notes example(2024.9.9)

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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

## Plasmid design

To produce recombinant tachypleus tridentatus clotting FG, we designed five plasmids in total for separate expression of its alpha and beta subunits as well as their co-expression.

For the E.Coli expression system, we designed two pET-28a plasmids with the optimized sequences of FG alpha and beta subunits respectively. In addition, we designed a pET-Duet1 plasmid with both sequences so that the subunits may bind more easily after expression. We hoped to accommodate this eukaryotic protein into a prokaryotic expression system, whether in selecting only the mature peptide sequences of the subunits from NCBI GenBank, or in performing codon optimization with NovoPro ExpOptimizer for elevated expression efficiency.

Since previous studies on recombinant FG production all employed eukaryotic expression hosts, we also designed two eukaryotic plasmids in case the protein failed to properly express through E.Coli. We optimized the sequences for sf9 insect cell expression hosts, then inserted them into two Pfastbac1 plasmids.

## Protein expression with E.Coli BL21

We transformed the pET-28a plasmids into E.Coli BL21(DE3) cells through heat shock, and inoculated the bacterial culture into kanamycin LB liquid medium at a concentration of 1:4. The bacterial culture was incubated at 37 ° C and 200 rpm for 1.5 hours before sample optical density was measured. When OD fell between 0.6-0.8, we induced protein expression with IPTG at a concentration of 1:1000.

However, during subsequent performance of SDS-PAGE, we discovered that the two subunits failed to express. No prominent protein bands were detected at 72 Kb and 37 Kb, corresponding to the alpha and beta subunits respectively. Our western blot assay similarly yielded no results.

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Image 1: SDS-PAGE & WB of failed expression

To investigate why neither subunits were expressed, we employed differents strain of competent cells, including Rosetta, Origami, and SHuffle, and repeated the previous experimental procedures. Target protein bands were obserservable at their corresponding molecular weights, showing ameliorated protein expression.

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Image 2: SDS-PAGE of successful expression

We then performed mass spectrometry to more accurately identify and quantify the expressed subunits. The protein bands were destained and dithiothreitol was added to break the disulfide bonds. We then enzymatically incised the protein into fragments of 700-3000 amino acids to facilitate mass spectrometry assay. The results indicated successful expression of both the alpha and beta subunits, though at a relatively low level. To boost production level, we further expressed the subunits with 5 liters of E.Coli. The mass expression and purification procedures are currently progress, and successful results are anticipated within a week.

## Experimental plans with sf9 insect cells

Since prokaryotic expression hosts may lack the protein modification mechanisms required for producing a fully functional horseshoe crab protein, we also conducted experiments with Spodoptera frugiperda 9 insect cells.

## BDG assay system construction

Upon acquiring the purified protein, we hope to test the activity of our protein, and the efficiency of recombinant Factor G in fungal infection detection. In the natural 1,3-BDG coagulation pathway, upon binding with fungal cell walls and detecting (1→3)-β-D-glucans (BDG), the α subunit of Factor G hydrolyses, releasing the β subunit, triggering a gelatinization/coagulation cascade, immobilizing fungal antigens[1]. In synthetic coagulation pathways, an indicator of this process is the hydrolysis of a synthetic chromogenic substrate, Boc-TGR-pNA(Nα-t-butyloxycarbonyl-L-threonyl-glycyl-arginine-p-nitroanilide), by the β subunit of Factor G, which releases yellow p-nitroaniline. We can then quantitatively measure the intensity of the color by spectrophotometrically measuring absorbance at 405nm wavelength.

We plan to perform a partial reconstruction of the pathway by first qualitatively testing both recombinant subunits separately. We will add Boc-TGR-pNA to our purified β subunit protein, and watch for qualitative changes in color that indicate a chromogenic reaction, and confirms the activity of our recombinant β subunit. We will then add (1→3)-β-D-glucans to purified α subunit protein, allowing it to react thoroughly, and running samples with SDS-PAGE to visualize whether the α subunit has undergone activation via proteolytic cleavage.

Subsequently, we plan on quantitatively testing the activity of our recombinant β subunit quantitatively measuring absorbance at 405nm, creating a standard curve, and comparing it to a standard curve created by current commercial G test kits. Finally, we will test the quantitative efficacy of our combined α and β subunits obtained by tandem expression. By comparing the efficiency of the BDG-assay-system using our recombinant FG protein with existing BDG test kits made from horseshoe crab protein, we hope to test the efficacy of our reconstructed fungal detection system alongside its ecological merits.

## References

- [1] Yamamoto Y, Kajiura H, Fukuchi H, Nishibu T, Fujiyama K, Kitagawa T. Reconstitution of (1→3)-β-D-glucans measurement system using recombinant Limulus polyphemus Factor G. Appl Microbiol Biotechnol. 2023 Dec;107(24):7463-7473. doi: 10.1007/s00253-023-12808-6. Epub 2023 Oct 26. PMID: 37882851.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Experiments

## Prokaryotic Experiment Protocol

Three competent cells we used:

**Rosetta(DE3)** chemically competent cell, **OrigamiB(DE3)** chemically competent cell and **SHuffle T7 Express lysY** chemically competent cell

## A. Transfection of Competent Cells:

1.Remove competent bacteria from -80°C and let thaw in ice (check with gentle flicks)

2. Add 2 µl of plasmid to each of the three types of competent cells and incubate on ice for 15 minutes.

3. Let sit on ice for 30 mins

4. Heat shock the cells at 42°C for 90 seconds

5. Place on ice for 2 mins

6. Add 1 mL of LB medium and place in shaking-culture (37°C, 200rpm) for 45 mins

7. Centrifuge at 4000 rpm for 3 minutes

8. Remove 800mL of clear liquid

9. Centrifuge at 4000 rpm for 3 minutes.

10. Resuspend in remaining LB and add solution to desired container of Kanamycin infused LB

11. Place in shaking-culture (37°C, 200rpm) for 24h

## B. Preparing Bacteria and Expression System:

1. Inoculate transformed E.Coli (1mL) into kanamycin LB medium (4mL)

2. Incubate at 37°C with shaking (209 rpm) for 1.5 hours

3. Measurement of optical density (OD) at 595nm

4. When OD=0.6-0.8, induce expression with IPTG (1 M, dissolved in ddH₂O) at 1:1000

5. Incubate at 16°C with shaking (220 rmp) for 20 hours.

## C. Sample Preparation:

1.After expression, centrifugate at 12000rpm for 1 min and remove clear liquid

2.Collect 400 µL of the bacterial culture

3.Resuspend using 80μL lysing buffer (adding 100mmol HEPES, 100mmol NaCl, 1mmol EDTA, 10% glycerinum and 0.5% Triton X-100) per tube

4.Add protease inhibitor

5.Sit on ice for 5-10 mins

6.Add 5XSDS loading buffer (20μL per tube)

7.Heat at 96°C for 10 mins

8.Load upper liquid as samples in SDS-PAGE

## D. SDS-PAGE:

1.**Preparation of Electrophoresis Tanks:** Clean and assemble the electrophoresis cassettes and tanks. Test the seal of the tanks by injecting water and observing the liquid level. Proceed if the liquid level shows minimal to no movement.

2.**Preparation of SDS-PAGE Gel:** Prepare the separation gel and pour 6mL into each glass plate.Seal the top with anhydrous ethanol. After the gel has polymerized, remove the ethanol and pour in the stacking gel, then insert the comb. Wait for the gel to set.

3.**3.Sample Loading and Electrophoresis:** Load 5 µl of protein ladder into the first well of each tank. Inject the prepared sample supernatants into the remaining wells. The proteins in the first tank will be stained using Coomassie Brilliant Blue, while the second tank will be used for Western blotting. Load 15 µl of sample into each well of the first tank and 10 µl into the second tank. Additionally, load 8 µg of lactoferrin in the last well of the first tank, and 5 µl in the last well of the second tank. Prepare the SDS-PAGE running buffer and fill the electrophoresis tank. Run the gel at 120V, for 45 mins.

## E. Mass Spectrum (MS):

1. Cut the selected gel strip with a surgical blade, place it in the EP tube, record the strip number and corresponding position, and cut the strip into 2 cubic millimeters

2. Add 500μL ultra-pure water (as the liquid level has not exceeded the volume of colloidal particles) and shock for 5 mins each time, and blot dry

3. Add acetonitrile (ACN)/50mM NH₄HCO₃=2:3 solution 500μL, ultrasonic decolorization for 5 mins or shock decolorization for 30 mins, and blot dry

4. Repeat step 3 until the blue fades

5. Add ACN 500μL to dehydrate, shake until the colloidal particles completely turn white, and blot dry (If the colloidal particles do not harden, repeat step 5)

6. Add 10mM dithiothreitol (DTT) (prepared with 10μL MDTT, 990μL 25mM NH₄HCO₃)100ul and bathe in water at 56℃ for 45 mins

7. Blot dry and quickly add 40mM iodoacetamide (IAM) (prepared with: 20μL 1M IAM, 480μL 25mM NH₄HCO₃) 100μL, place in dark for 45mins

8. Wash with 50%ACN solution and ACN successively, dehydrate ACN until the colloidal particles are completely white (standard reference step 5), and blot dry

9. Dilute the 0.1μg/μL enzyme storage liquid 20 times with 25mM NH₄HCO₃. The amount of enzyme liquid added to each EP tube is subject to complete wetting of the colloidal particles, slightly centrifuge, so that the enzyme liquid is fully in contact with the colloidal particles. Place it in 4℃ or on ice for 5 mins, until the solution is fully absorbed by the rubber block, absorb the excess enzyme liquid. Add 25mM NH₄HCO₃ 45μL (immersed in colloidal particles and more than 30μL) in a 37℃ water bath, digest overnight

10. Add 10% formic acid (FA) to terminate the reaction, oscillate and mix, centrifuge, PH

11. Remove the solution and transfer it to a new EP tube, label it, add 60%ACN to the tube with colloidal particles for ultrasonic extraction twice, 10 mins each time, combine the solution three times, vacuum dry the solution, add 0.1%FA aqueous solution 20μL to dissolve the polypeptide

## F. Purification:

1.Attach the Ni2+ affinity column to the pump head and wash 5 column volumes with a low concentration imidazole salt buffer.

2. Put the protein expressing bacteria solution into a high-speed centrifuge for centrifugation, 5000 rpm, 4 °C, 10 minutes, collect the bacteria, frozen at -20 °C or -80 °C for later use.

3.Resusbend the bacteria with low concentration imidazole buffer.

4.Under the condition of ice bath, release the expressed protein by pressure crusher or ultrasonic crusher. If the bacterial solution is still cloudy after ultrasound, it is necessary to increase the power appropriately and extend the ultrasonic time.

5.Place the suspension in an ultra-high-speed centrifuge at 4°C, 18000 rpm, for 25 minutes, to separate the upper protein albumen and precipitate.

6.SDS-PAGE electrophoresis detection: Determine whether the protein is expressed in soluble or inclusion body (precipitation).

7. Connect the Ni2+ affinity column to the pump head, and the protein cracking solution is passed through the Ni2+ affinity column at a slow and constant speed at 4 °C.

8.Use low concentration imidazole buffer (10 mM imidazole 2-5 mL/min) flow through the Ni2+ affinity column, about 5 column volumes.

9. Perform phase elution with imidazole buffers containing 50, 100, 200, 300 and 400 mM, respectively, with flow rates of 2-5mL/min. Collect eluting peaks at each stage, and each concentration of imidazole buffer flowed through 2 column volumes.

10. Collect protein samples and use SDS-PAGE to detect the molecular weight and purity of the fusion protein.

11.Use ddH₂O to wash 5 column volumes of Ni2+ affinity columns, and then wash 3 column volumes of Ni2+ affinity columns with 20% ethanol at a flow rate of 2-5 mL/min. The columns were always kept in a low temperature environment.

## Eukaryotic Experimental Protocol

## A. Culture Insect Cells:

For the culture of Sf9 insect cells, we employed the serum-free and protein-free medium Sf-900™ II SFM. Cells were maintained at 27-28°C in shaking flasks(120-140rmp) until they reached the required cell density (1-2 \* 10⁶ cells/mL).

## B. Transfection of Sf9 Cells:

We mixed 50 µL of ExpiFectamine™ Sf reagent with 100 µl of DNA and incubated for 5-10 minutes at room temperature. We then added the DNA-reagent mix to 2 \* 10⁶ cells/mL of Sf9 cells and incubated for 5 hours at 27°C.

## C. Baculovirus Production:

After the transfection procedure, the cells were cultured at 27°C with shaking for 72 hours. We then harvested the P0 virus stock through centrifugation (1,000 \* g for 5 minutes) and stored it at 4°C for immediate use or at -80°C for long-term storage.

## D. Amplification of Baculovirus

The P0 virus stock was inoculated into 50 mL of Sf9 cells (2 \* 10⁶ cells/mL) and incubated at 27°C with shaking (120-140 rpm) for 72 hours. We then harvested the P1 virus stock with centrifugation as previously described.

## E. Large-Scale Protein Expression

We inoculated Sf9 cells at 2 \* 10⁶ cells/mL with the P1 virus stock at a multiplicity of infection (MOI) of 0.01-1 and incubated at 27°C with shaking (120-140 rpm) for 48-72 hours until signs of infection were observed (e.g., enlarged size, reduced viability). We then harvested the cells by centrifugation at 1,000 \* g for 5 minutes.

## F. Protein Purification

The cell pellets were taken and resuspended in lysis buffer. Cells were lysed with a sonicator and centrifuged at 20,000 \*g for 30 minutes.

We used nickel affinity chromatography for purification of the his-tagged recombinant proteins. The column was first loaded with 2 mL of equilibration buffer (50 mM sodium phosphate, 300 mM NaCl, pH 8.0). We then added the supernatant onto the equilibrated Ni-NTA column and incubated for 1 hour at 4°C with gentle mixing to ensure binding between the His-tagged protein and the resin.

To remove non-specifically bound proteins, we washed the column with 10 mL of wash buffer (50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0) and discarded the flow-through. To collect the bound target protein, we eluted the column with 2-3 column volumes of elution buffer (50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 8.0). We would then confirm the presence of our recombinant protein through western-blot or mass spectrometry.

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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# Results

Through mass spectrometry, we identified peptides that cover 44.91% of the α-subunit protein(697 aa) sequence, indicating that the protein has been expressed and identified to a certain extent in the expression system. Specifically, we observed 124 peptide-spectrum matches (PSMs), which provide a solid foundation for the identification of the α-subunit protein. In addition, there are 28 unique peptides matched to the α-subunit protein, and these unique peptides are a sign of identifying a certain protein, and their identification further strengthens our confidence in the identification results of the α-subunit protein. The theoretical molecular weight of the α-subunit protein is 77.9 kDa, and the protein matching score Score Sequest HT is 311.53, this high score indicates a high credibility of protein matching.

The expression and mass spectrometry analysis results of the β-subunit protein(322 aa) in the E.Coli expression system are also encouraging. Mass spectrometry analysis shows that the identified peptides cover 63.66% of the β-subunit protein sequence, and this high coverage indicates that the expression and identification efficiency of the β-subunit protein in the expression system are relatively high. In addition, we recorded 146 peptide-spectrum matches (PSMs), which further confirm the successful identification of the β-subunit protein. It is worth noting that there are 15 unique peptides matched to the β-subunit protein, and the identification of these unique peptides provides strong evidence for the identification of the β-subunit protein. The theoretical molecular weight of the β-subunit protein is 35 kDa, and the protein matching score Score Sequest HT is 381.62, this high score also indicates a high credibility of protein matching.

The molecular weight of β-subunit protein is relatively smaller, but its identification coverage is higher than α-subunit, which may indicate its expression efficiency in the expression system.

![](https://static.igem.wiki/teams/5173/result.jpg)

Table 1: MS result

![](https://static.igem.wiki/teams/5173/engineering/sds3.jpg)

Image 1: SDSZ-PAGE result

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