iGEM 2024 — Team hk-united

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{% block title %}Engineering Success{% endblock %}
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## Design 1

![](https://static.igem.wiki/teams/5063/engineering/puc18-with-tp1-p5.png)

(fig a. our recombinant plasmid design, utilizing the pUC18 vector.)

Our objective is to biosynthesize the fusion protein TP1\_P5 in E.coli under optimal culture conditions. To achieve this, we have designed a recombinant plasmid specifically tailored to facilitate the expression of our target protein.

The plasmid incorporates a T7 promoter, which ensures robust transcription of our insertion when induced. Following the promoter, we included a ribosome binding site (RBS) to enhance translation efficiency. This design features multiple start codons, allowing flexible initiation of translation based on the cellular environment.

The fusion protein sequence is strategically positioned downstream of the RBS, followed by a TEV cleavage site for the post-expression processing of our fusion protein, enabling efficient cleavage to release TP1\_P5 from any fusion partners or tags. Additionally, we have incorporated a 6x histidine tag (His-tag) to facilitate the purification process via metal affinity chromatography, which allows for specific binding to nickel ions during purification.

The construct ends with stop codons and a double terminator to ensure proper termination of transcription.

## Build 1

We designed our gene construct based on the pUC-18 vector, and we inserted our recombinant plasmid into two types of E. coli. , DH5 Alpha and BL21, and we purified the proteins to prove that TP1P5 is present.

### Transformation

The synthetic gene block containing our genes of interest (TP1 and P5) was transformed into DH5 Alpha and BL21. The two types of cells were then frozen and prepared for cell culture.

### Cell Culture & Protein Purification

50μL of the cell samples were spread on agar gel and incubated at 37°C. A colony of the cells was then selected and used for cell culture. Thus, we have a sufficient amount of cells containing the plasmid containing our fusion protein.

The cells were then underwent protein purification to obtain a pure sample of our protein to confirm the presence of TP1P5 without contamination and interference from other proteins.

## Test 1

We conducted gel electrophoresis alongside the SDS-PAGE method to prove the existence of our proteins. These methods could provide us the length of both the DNA and the protein. By comparing the position of the protein of interest to the protein ladder, we could deduce the size of the protein. If the position of our protein relative to the protein ladder corresponds to the theoretical size of our plasmid, then the experiment would be deemed successful.

## Learn 1

We believe that the expression of the protein in the bacteria is insufficient, according to the faint colour of our protein band in the gel demonstrated. Henceforth, we have to navigate a method that can increase the production of our fusion protein inside E. coli, such as incorporating another protein strain inside of our plasmid that can increase the synthesis of our gene of interest.

## Design 2

We subsequently developed new versions of the recombinant plasmid that incorporate the DSBA domain. DSBA is a disulfide bond isomerase that catalyses intrachain disulfide bond formation, which is essential for the proper folding of proteins that contain disulfide bonds. By integrating the DSBA sequence into our plasmid. We aim to enhance the expression and stability of our target fusion protein, TP1\_P5, in the E. coli BL21 strain, and for that, we have switched to another transcription vector, pET-21, designed for expression from bacterial translation signals carried within a cloned insert.

![](https://static.igem.wiki/teams/5063/engineering/pet-21-dsba.png)

(fig b. the separately expressed DSBA plasmid, using the pET-21 vector)

Aside from that, we have also considered the varying severity and scenarios of the cardiovascular diseases and to prevent overdosing, we decided to express TP1 and P5 separately. Similarly, we have incorporated a 6x His-Tag to facilitate the purification process as round one.

Ginsentide TP1 demonstrates a multifaceted approach to promoting cardiovascular health through its activation of the PI3K-Akt pathway and subsequent effects on nitric oxide production, vascular relaxation, and platelet aggregation inhibition.

Lupin peptide P5 has emerged as a promising candidate in cholesterol management, exhibiting statin-like properties through its competitive inhibition of HMG-CoA reductase. By targeting this crucial enzyme in the mevalonate pathway, Lupin peptide P5 plays a significant role in reducing cholesterol production and enhancing the uptake of low-density lipoprotein (LDL) particles.

When our fusion protein is in action, it will accumulate in certain regions of the afflicted blood vessel. The concentration of our in-action fusion protein would vary depending on the severity of the damage of the blood vessel. The more concentrated our proteins are, indicating the more severe the infection is. This is where our newly implemented green fluorescent protein (GFP), and a smaller luminescent probe (miRFP670nano) comes into play, where it acts as an alternative diagnostic tool, indicating the locations of our fusion proteins, and we can subsequently tell the concentration of our fusion protein, alongside the severity of the situation. This can demonstrate the current situation of the blood vessel, and professionals could judge and decide whether immediate surgery should be carried out, while preventing the situation from worsening.

Through the regulation of tp1 and p5 dosages in our product, we can offer personalised medicine for patients with different extents of endothelial injury platelet aggregation and LDL level; and with the addition of GFP and the probe, we could provide a more affordable alternative of diagnostic measures for both professionals and laymen.

![](https://static.igem.wiki/teams/5063/engineering/pet-21-tp1p5-gfp.png)

(fig c. Our revised recombinant plasmid featuring GFP, utilizing the pET-21 vector)

![](https://static.igem.wiki/teams/5063/engineering/pet-21-tp1p5-mirfp670nano.png)

(fig d. Our revised recombinant plasmid featuring miRFPnano, utilizing the pET-21 vector)

To maximise the effectiveness of this approach, we will first transform the BL21 strain with the plasmid containing the DSBA part. This initial transformation will enable the bacteria to express DSBA, increasing the availability of this important chaperone protein within the cells. The presence of DSBA is expected to facilitate the proper folding of our target protein, thereby improving the yield of correctly folded TP1\_P5.

After establishing a stable BL21 strain with DSBA expression, we will proceed with a second round of transformation to introduce our recombinant plasmid containing TP1\_P5. This two-step transformation strategy not only ensures a higher concentration of DSBA in the bacterial system but also allows us to create a more favourable environment for the expression of disulfide-bonded proteins.

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# Polymerase Chain Reaction(PCR)

The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification (1). Taq DNA Polymerase is an enzyme widely used in PCR (2). The following guidelines are provided to ensure successful PCR using New England Biolabs’ Taq 2X Master Mix. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure, low template concentrations, or
amplicons greater than 5 kb may require further optimization.

## Reaction setup

all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95°C).

## Component cell:

25 µl reaction 50 µl reaction Final Conc.

10 µM Forward Primer 0.5 μl 1 μl 0.2 µM (0.05–1 µM)

10 µM Reverse Primer 0.5μl 1 μl 0.2 µM (0.05–1 µM)

Template DNA variable variable 1,000 ng

Taq 2X Master Mix 12.5μl 25 μl 1X

Nuclease-free water to 25 µl to 50 µl

Transfer PCR tubes from ice to a PCR machine with the block

preheated to 95°C and begin thermocycling

## Thermocycling conditions for a routine PCR:

STEP TEMP TIME

Initial Denaturation 95°C 30 seconds

30 Cycles 95°C

45-68°C

68°C 15-30 seconds

15-60 seconds

1 minute per kb

Final Extension 68°C 5 minutes

Hold 4-10°C

## Plasmid Expression And Purification

#### Plasmid Miniprep Kit

The Plasmid Miniprep Kit is a rapid and reliable method for the purification of high quality plasmid DNA. This method employs standard cell resuspension, alkaline lysis, and neutralization steps, with the additional benefit of color indicators at certain steps to easily monitor completion. Unique wash buffers ensure salts, proteins, RNA and other cellular components are removed, allowing low-volume elution of concentrated, highly pure DNA. Elution in as little as 30 μl provides concentrated DNA for use in downstream applications, such as restriction digests, DNA sequencing, PCR and other enzymatic manipulations.

## PLASMID REPLICON COPY NUMBER CLASSIFICATION

pUC and its derivatives pMB1 >75 High copy

pBR322 and its derivatives pMB1 15–20 Low copy

pACYC and its derivatives p15A 10–12 Low copy

pSC101 pSC101 -5

Buffer Preparation:

Add ethanol to Monarch Plasmid Wash Buffer 2 prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch Plasmid Wash Buffer 2).

For 50-prep kit add 24 ml of ethanol to 6 ml of Monarch Plasmid Wash Buffer 2
For 250-prep kit add 120 ml of ethanol to 30 ml of Monarch Plasmid Wash Buffer 2
Protocol:
All centrifugation steps should be carried out at 16,000 x g

1. .Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended.
2. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Incubate for one minute.
3. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until colour is uniformly yellow and a precipitate forms.Incubate for 2 minutes.
4. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
5. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute.
6. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
7. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through
8. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

# Cell Lysis

## E. coli Lysis Reagent

NEBExpress E. coli Lysis Reagent is a chemical lysis solution composed of a proprietary mix of non-ionic and zwitterionic detergents and Tris-based buffer. It allows disruption of E. coli cells without denaturing soluble proteins.

#### Protocol

1. If monitoring cell density by OD600, record final OD readings prior to harvesting.
2. Harvest cells by centrifugation at 16,000 x g for 10 minutes. For larger volumes, centrifuge for ≥30 minutes especially if at ≤10,000 x g. Discard the medium and, if necessary, weigh the wet cell pellet.
3. Store the pellet at -20°C or -80°C or process immediately.
4. Resuspend the cell pellet in NEBExpress E. coli Lysis Reagent by pipetting or vortexing briefly until the suspension is homogenous:
5. Use 0.025 - 0.075 mL of NEBExpress E. coli Lysis Reagent for every 1 UOD600 harvested. To calculate the UOD600, multiply the volume harvested by the OD600 reading.
   For example, a 5 mL culture harvested at OD600 1 gives 5 mL x 1.0 = 5 UOD600. In this example, 0.125 – 0.375 mL Lysis Reagent is required to lyse efficiently.
6. If harvested cells are weighed, use 5 mL of NEBExpress E. coli Lysis Reagent per 1 gram of cells.
7. Incubate the resuspended cells at room temperature for 10 - 20 min with gentle shaking, gentle rotation, or swirling. Lysis is usually visible with a clearance of the suspension.
8. Centrifuge the lysate at 16,000 x g for 10 min at 4°C to pellet the insoluble material and cell debris (30 min or longer for large volumes and lower speed).
9. Carefully transfer the supernatant into a sterile container for analysis or purification. This soluble fraction can be stored at 4°C for a few hours or -20°C or -80°C for longer term storage.
10. If needed, resuspend the insoluble pellet in 50 mM Tris-HCl pH 7.5 or any desired buffer for analysis or purification of inclusion bodies.

# Protein PurificationNi Spin Columns Spin columns containing an affinity matrix for the small-scale isolation and purification of polyhistidine-tagged (His-tagged) fusion proteins. Immobilized Metal Affinity Chromatography (IMAC) purifications employing NEBExpress® Ni Spin columns can be performed under native or denaturing conditions, yielding highly pure target in a single protein purification step. This enables screening of expression conditions and streamlines the functional and structural characterization of the target protein. Protocol 1.Since the protein produced in BL21 matches the estimated kDa of our fusion protein, the plasmid insertion is proved to be successful, and our desired protein Gel electrophoresis Gel electrophoresis is a laboratory method used to seperate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be seperated are pushed by an electric field through a gel that contains small pores Protocol 1. Preparation of Agarose Gel 2. Dilute 50X TAE Buffer to 1X TAE buffer. 3. Add desired amount of ultra-pure agarose to 1X TAE buffer in a flask. 4. Microwave solution for 1 min. Microwave for longer if there is undissolved agarose. 5. Allow the solution to cool for 1 min. 6. Pour the solution inro the gel tray. Insert the comb into the top of the gel. 7. after 15 mins, 3μL GelGreen® Nucleic Acid Gel Stain was added into the gel solution. 8. Allow the gel to solidify another 15mins. 9. Running gel electrophoresis 10. Once the gel has solidified, carefully remove the comb by pulling straight up. 11. Prepare the samples by adding 6X loading buffer to each. 12. Load 15μL of DNA ladder into one of the lane. 13. Load samples into wells. Avoid bubbles. 14. Remove the tray with the gel and image with UV High Efficiency Transformation using both in DH5-alpha and BL21(DE3) High Efficiency Transformation using both in DH5-alpha and BL21(DE3) In transformation, cell are made competent (able to take up exogenous DNA) by treatment with divalent cations such as calcium chloride, which make the bacterial cell wall more permeable to DNA. Heat shock is used to temporarily form pores in the cell membrane, allowing transfer of the exogenous DNA into the cell. Protocol 1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. 2. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice. 3. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. 4. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex. 5. Place the mixture on ice for 30 minutes. Do not mix. 6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix. 7. Place on ice for 5 minutes. Do not mix. 8. Pipette 950 µl of room temperature SOC into the mixture. 9. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. 10. Warm selection plates to 37°C. 11. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC. 12. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. 13. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours. Protein Expression Using BL21(DE3)Protocol 1. Transform expression plasmid into BL21(DE3). 2. Plate on antibiotic selection plates and incubate overnight at 37°C. 3. Resuspend a single colony in 10 ml liquid culture with antibiotic. I 4. ncubate at 37°C until OD600 reaches 0.4–0.8. 5. Induce with 4 or 40 µl of a 100 mM stock of IPTG (final concentration of 40 or 400 µM) and induce for 3 to 5 hours at 37°C. 6. Check for expression either by Coomassie stained protein gel, Western Blot or activity assay. 7. Check expression in both the total cell extract (soluble + insoluble) and the soluble fraction only. SDS-PAGEProtocol 1. Place the the gel in the SDS-PAGE stand 2. Place the stand with the gel in the SDS-PAGE apparatus bath 3. fill the space between the gels with running buffer 4. Take off the combs of the gel (take it out gently) 5. Load the sample to each well 6. Do not use the wells at the left and right end of the gel 7. fill the SDS-PAGE apparatus bath with running buffer to the bottom of the gel 8. Place the cover of the SDS-PAGE 9. Plug the cables to the SDS-PAGE apparatus power source 10. Turn of the SDS-PAGE power source 11. Set to constant voltage 12. Use 200 13. Press the Run button to start the electrophoresis 14. The voltage increases to 200 V from 0 V 15. For 4% staking gel and 12.5 % separating gel the current reaches 50-60 mA and the power 10 -12 W 16. The current the the power decreases in time but not reach zero (if it reaches zero see the troubleshoot) 17. For the state type of gel it takes 35 - 40 min

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# Results

## Transformation of TP1\_P5\_GFP recombinant plasmid

We successfully produced recombinant plasmid, with 6.4 KBP (6389 DNA base pairs). By the process of transformation, we have successfully bioengineered a kind of bacteria, which can produce the protein that we want. The following picture indicates that the incubated bacteria, which was on an agar plate and it was stored for further usage. We intentionally added antibiotics in the agar plate to test whether the bacteria can be incubated successfully. If it can’t be killed by those specific antibiotics, it means that the bacteria is the specific bacteria with our recombinant plasmid.

To prove that the modified bacteria has our designed gene, we used gel electrophoresis to check whether the bacteria contains our gene or not. In the picture below, we can see the band of our bacteria at the position around 6.4 KBP of the ladder, which is exactly the length of our gene. This indicates that the bacteria contains our designed gene.

![](https://static.igem.wiki/teams/5063/result/result1.jpg)

## Produce our recombinant protein

To prove that the modified bacteria has GFP (green fluorescent protein), we can see the green fluorescence in the incubated BL21 bacteria under a blue light source in the picture below.

![](https://static.igem.wiki/teams/5063/result/results2.jpg)

(IPTG is used to induce the expression of the gene in BL21)

Since this solution shows fluorescence, we extracted the protein by cell lysis and underwent protein purification. To prove that the bacteria can produce the protein we want, we test the extract by SDS-page.

![](https://static.igem.wiki/teams/5063/result/result3.jpg)
![](https://static.igem.wiki/teams/5063/result/result4.jpg)

The band in the range 34 kDa proves the expression of TP1\_P5\_GFP

Since the protein produced in BL21 matches the estimated kDa of our fusion protein, the plasmid insertion is proved to be successful, and our desired protein can be produced.

![](https://static.igem.wiki/teams/5063/result/34-kda.png)

The band in the range 34 kDa proves the expression of TP1\_P5\_GFP

Since the protein produced in BL21 matches the estimated kDa of our fusion protein, the plasmid insertion is proved to be successful, and our desired protein

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