iGEM 2025 — Team hia-taiwan

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![Top Image](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/lightbluetitlebg.png)

# Notebook

Table of Content

| Date | Entry Title | Group | Notebook | Page |
| --- | --- | --- | --- | --- |
| Aug 26, 2025 | PCR of BLIP-I | A | 1 | 1 |
| Dilution of 10× TAE Buffer to 1× and Autoclaving | A | 1 | 2 |
| Aug 29, 2025 | Restriction Digestion of pSB1C3 | A | 1 | 2 |
| Preparation of LB agar and LB broth | B | 1 | 3 |
| Rehydration of E.coli S17-1 | B | 1 | 3 |
| Aug 30, 2025 | Resuspension of BLIP-II, PCR of BLIP-II, and Restriction Digestion of BLIP-I & pSB1C3 | A | 1 | 4 |
| Preparation of S17-1 glycerol stock | B | 1 | 4 |
| Sep 2, 2025 | Gel electrophoresis of products from 8/30 (BLIP-II PCR, BLIP-I digest, pSB1C3 digest) | A | 2 | 1 |
| Subculture of E.coli | B | 2 | 2 |
| Sep 4, 2025 | Reviving E. coli DH5α from Glycerol Stock – Plate (Part 1) | B | 2 | 2 |
| Preparation of S17-1 Glycerol Stock (from Plate) - Failed Attempt | B | 2 | 3 |
| Sep 5, 2025 | Gel electrophoresis of BLIP-I RD, pSB1C3 RD, and BLIP-II PCR | A | 2 | 4 |
| Reviving E. coli DH5α from Glycerol Stock – Broth (part 2) | B | 2 | 5 |
| Inoculation of S17-1 into LB Broth | B | 2 | 5 |
| Sep 6, 2025 | Gel extraction of products made on 9/5, PCR and restriction digestion of BLIP-I and BLIP-II, gel run of both | A | 2 | 6 |
| Preparation of S17-1 Glycerol Stock (from Broth) | B | 2 | 7 |
| Preparation of Antibiotic Plates (Amp + Chl) | B | 2 | 7 |
| Sep 8, 2025 | Ligation and gel run of BLIP-I (PCR product made on 9/6) | A | 3 | 1 |
| Resuspension of BBa\_J435300 (Distribution Kit DNA) | B | 3 | 1 |
| Sep 10, 2025 | Gel Extraction of BLIP-I (PCR product from 9/6) | A | 3 | 2 |
| Heat-Shock Transformation of BBa\_J435300 into DH5α | B | 3 | 2 |
| Sep 12, 2025 | Ligation of BLIP-I and BLIP-II (T4 DNA ligase) | A | 3 | 3 |
| Overnight Culture of S17-1 Subculture | B | 3 | 3 |
| Sep 13, 2025 | Preparation of Competent Cells (S17-1, CaCl2 method) | A&B | 3 | 4 |
| Sep 15, 2025 | Subculture of E. coli S17-1 (from 08/29 rehydration plate) | B | 4 | 1 |
| Sep 16, 2025 | Preparation of Competent Cells (CaCl₂ method, Attempt 1) | A | 4 | 2 |
| Preparation of LB Agar and LB Broth (no antibiotics & Amp) | B | 4 | 2 |
| Sep 17, 2025 | PCR of BLIP-I | A | 4 | 3 |
| Preparation of Competent Cells (CaCl₂ method, Retry) | B | 4 | 3 |
| Sep 18, 2025 | PCR of BLIP-II | A | 4 | 4 |
| Resuspension of pSB1C3-mCherry (Distribution Kit DNA - O11) | B | 4 | 4 |
| Heatshock transformation of (DH5α + mCherry, pSB1C3) | B | 4 | 4 |
| Sep 19, 2025 | BLIP-I gel run | A | 4 | 5 |
| PCR of BLIP-II | A | 4 | 6 |
| BLIP-I purification | A | 4 | 6 |
| Heat Shock Transformation (DH5α + BBa\_J435300) | B | 4 | 6 |
| Plating of Pinky (DH5α + mCherry, pSB1C3) | B | 4 | 6 |
| Sep 20, 2025 | Gel Electrophoresis of BLIP-I & BLIP-II | A | 4 | 7 |
| Plating | B | 4 | 8 |
| Miniprep of Pinky Plasmid (pSB1C3 backbone) | B | 4 | 8 |
| Preparation of Selective Agar Plate | B | 4 | 9 |
| Sep 22, 2025 | Restriction Digestion of BLIP-I, BLIP-II, and pSB1C3 | A | 5 | 1 |
| Sep 23, 2025 | Gel Electrophoresis of 9/22 Restriction Digestion Products (BLIP-I, BLIP-II, pSB1C3) | A | 5 | 2–3 |
| Resuspension of BBa\_J435500 (Distribution Kit DNA - O6) | B | 5 | 3 |
| Heat Shock Transformation (BBa\_J435500 + DH5α) | B | 5 | 4 |
| Sep 24, 2025 | Restriction Digestion and Gel Electrophoresis of BLIP-I, BLIP-II, and pSB1C3 | A | 5 | 5 |
| Subculture and Liquid Culture of DH5α + BBa\_J435500 (no antibiotic) | B | 5 | 6 |
| Sep 25, 2025 | PCR of BLIP-I | A | 5 | 7 |
| Sep 26, 2025 | Gel run of samples from 9/25 and gel extraction following the new method | A | 5 | 8 |
| Overnight Culture & Glycerol Stock Preparation | B | 5 | 9 |
| Sep 27, 2025 | Run gel for purified BLIP-I, BLIP-II DNA, restriction digestion of BLIP-I, BLIP-II, and pSB1C3 | A | 5 | 10–11 |
| Miniprep & Glycerol Stock Preparation | B | 5 | 11 |
| Sep 30, 2025 | Gel extraction of digested product (new method) and Ligation | A | 6 | 1 |
| Resuspension & Heat Shock Transformation of BBa\_J435300 (Well O9) | B | 6 | 2 |
| Oct 1, 2025 | Test OD for transformation products, Resuspend primers, PCR for BLIP-II | B | 6 | 3 |
| Transformation of BLIP-I and BLIP-II into pSB1C3 (DH5α and S17-1) | B | 6 | 4 |
| Oct 2, 2025 | PCR for BLIP-I and BLIP-II; Growth of Single Colony DH5α | B | 6 | 5 |
| Oct 3, 2025 | PCR of BLIP-I and Gel run of PCR products | A | 6 | 6 |
| Subculture of S17-1 and DH5α + BLIP-I (broth) | B | 6 | 7 |
| Oct 4, 2025 | Preparation of Streptomycin LB Broth & Competent S17-1 Cells | B | 6 | 8 |
| Transformation of pSB1C3 + BLIP-I / BLIP-II into S17-1 | B | 6 | 8 |
| Miniprep of pSB1C3 + BLIP-I, BLIP-II, and BBa\_J435320 from DH5α | B | 6 | 9 |
| Oct 5, 2025 | Overnight Culture of BLIP-I and BLIP-II | A | 6 | 10 |
| Oct 6, 2025 | Conjugation of S17-1 (BLIP-I & BLIP-II) with DH5α | A&B | 7 | 1-2 |
| Oct 7, 2025 | Collected data on solid and liquid conjugation | A&B | 7 | 2-3 |

AUGUST

[WEEK 1 (2025/08/26 ~ 2025/08/30)](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/n1.pdf)

SEPTEMBER

[WEEK 2 (2025/09/01 ~ 2025/09/07)](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/n2.pdf)
[WEEK 3 (2025/09/08 ~ 2025/09/14)](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/n3.pdf)
[WEEK 4 (2025/09/15 ~ 2025/09/21)](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/n4.pdf)
[WEEK 5 (2025/09/22 ~ 2025/09/28)](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/n5.pdf)

OCTOBER

[WEEK 6 (2025/09/29 ~ 2025/10/05)](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/n6.pdf)
[WEEK 7 (2025/10/06 ~ 2025/10/08)](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/newnewn7.pdf)

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[SUMMARY](engineering)
[PROJECT DESIGN](#project_design)
[WET LAB](#wet_lab)
[DRY LAB](#dry_lab)
[HUMAN PRACTICES](#hp)

![Top Image](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/darkbluetitlebg.png)

# Engineering

## Engineering

**Project Design:**
Explore how PlasMission progressed through three major design plans and innovative ways to overcome technical barriers, initiate validation processes, and reinforce hypotheses. Witness how we internalize conjugation technologies into stopping beta-lactamase bacteria and shape the ultimate plan for conjugation and reliable validation of both successful conjugation and efficacy of BLIP.

**Wet Lab:**
Find out how PlasMission navigates through a total of four transformation steps in constructing a complete genetic circuit, each carefully designed to assemble functional modules with precision and compatibility. See how we employ the RP4 conjugation mechanism to facilitate horizontal gene transfer, enabling the donor bacteria carrying the BLIP gene to successfully deliver the target gene into recipient bacteria harboring ampicillin resistance. This process not only demonstrates controlled genetic exchange but also validates our conjugation system as a reliable platform for plasmid mobilization.

**Dry Lab:**
Simulate models of bacterial conjugation and the interaction between BLIP and β-lactamase. Our team built two computational models to understand how our BLIP systems fight antibiotic resistance. The first model focused on how plasmids spread between bacteria through conjugation, helping us estimate how efficiently the BLIP genes could transfer. The second model, created in COPASI, explored how BLIP interacts wit β-lactamase to block its activity and protect antibiotics. This allows us to compare the efficiency BLIP-I and BLIP-II degrading BLA. Together, these models helped us predict system performance even when lab experiments couldn’t be completed on time, giving us a clearer picture of how our experiment will and should work.

**Human Practices:**
Take a look at how PlasMission integrates synthetic biology, antibiotic resistance, and other applications into education and outreaches. A total of 6 interviews, 13 physical outreaches, and 3 education activities. In between each we make mistakes, reflect, and rebuild each interview and outreach methods.

## Project Design Codon Optimization Cycle 1

**Design**
Explore how For BLIP expression and validation purposes, our insertion gene sequence is composed of RP4 oriT promoter, two RBS, BLIP-I/BLIP-II gene expression, fluorescent protein, and a terminator. We used the screen sequence function provided by Integrated DNA Technology (IDT) PlasMission progressed through three major design plans and innovative ways to overcome technical barriers, initiate validation processes, and reinforce hypotheses. Witness how we internalize conjugation technologies into stopping beta-lactamase bacteria and shape the ultimate plan for conjugation and reliable validation of both successful conjugation and efficacy of BLIP.

**Build**
Once everything was put together, the first version of codons had a complexity score of up to 78.1 for BLIP-I, and 87.22 for BLIP-II. We adjusted codons according to IDT’s optimizing guidelines. The first version of optimization was done with codon optimization tools provided by IDT, Benchling, and Snapgene.

**Test**
After optimizing all the lines that either had repeated sequences that were too long, or a too high number of GC content with codon optimizing tools listed above, the amino acids appeared to be different from the initial protein sequence. This process was repeated several times, all resulting in different amino acid sequences, different from our targeted sequence.

**Learn**
In this process we learned and adapted from only using codon optimization tools, to adjusting some parts of the gene sequence by manually. Although this process was more complicated and had a relatively higher error rate, after doing the same process of optimization for three times, we managed to get a result that was the same and authenticated.

## Project Design Validation Method Cycle 1/2

**Design**
To validate the successful conjugation and transfer of the BLIP gene, we designed three complementary approaches. First, we used fluorescent markers, embedding green and red fluorescent proteins in the recipient and donor plasmids, respectively—successful conjugation would produce transconjugants expressing both colors. Second, we applied an antibiotic resistance assay, using selective plates to distinguish donors, recipients, and transconjugants based on their survival patterns. Third, we performed PCR amplification targeting BLIP-I and BLIP-II sequences to confirm their presence in transconjugant colonies. After further research, we refined our validation strategy by introducing optimized plasmid designs to enhance efficiency and reliability.

**Build**
Through this planning process, we identified potential challenges and limitations in our validation setup during later times when we received our distribution kit. There were no available pSB1K3 in the distribution kit, making it difficult for us to carry out the plan.

**Test**
Additionally, although the current validation method provides us with roughly estimated efficacy of BLIP, there is no direct proof of BLIP actually working against beta-lactam resistant bacteria.
Furthermore, the first validation method required a confocal for both colors to show at the same time, but we do not have access to it.

It was also predictable that the survival rate of the transconjugant bacteria to survive dual antibiotic plate might be too low for effective validation.

**Learn**
To conclude, this first validation would not be an ideal way to confirm the efficacy of BLIP and successful conjugation. The survival of the transconjugant on Kanamycin and Chloramphenicol plate is too unstable to provide reliable results and is unable to prove the functionality of BLIP.

## Project Design Validation Method 2/2

**Design**
After the first cycle, we refined our system to directly demonstrate the efficacy of BLIP against ampicillin-resistant bacteria. Initially, we believed that working with ampicillin-resistant strains might not be permitted under the iGEM High School Safety White List, making validation challenging. However, after researching iGEM safety regulations and consulting biosafety experts, we confirmed that ampicillin resistance falls under β-lactamase resistance (Ambler Class A) — a category that is safe for BSL-1 high school teams.
This confirmation allowed us to proceed with a scientifically valid and safety-compliant design that effectively showcases BLIP's inhibitory potential.

**Build**
We established a new validation workflow to assess conjugation and inhibition more effectively:
- Continued using PCR and gel electrophoresis for molecular confirmation.
- Implemented cross-plating of donor and recipient cells on three antibiotic conditions: Chloramphenicol (Ch), Ampicillin (Amp), and Dual Ch + Amp. These conditions allowed us to compare survival patterns and evaluate BLIP’s inhibitory function.
- Introduced color-based differentiation for better visualization: Red colonies (mCherry) = donor strain, blue colonies (lacZ/X-gal reaction) = recipient strain, purple/mixed colonies = transconjugants carrying both markers. This combination of antibiotic selection and phenotypic color screening enabled precise differentiation among donor, recipient, and transconjugant populations.

**Test**
The cross-plating validation method successfully distinguished all cell types under selective conditions. Although IPTG did not arrive in time, we proceeded with blue-white screening using X-gal alone. Because the recipient plasmid (BBa\_J435320) carries a leaky lac operon, some basal expression of lacZ occurs even without induction. As a result, the recipient and transconjugant colonies displayed a weak blue coloration, confirming that the color-based differentiation still functioned as expected.

For BLIP-II, no colonies appeared on the Amp plates, demonstrating complete inhibition of β-lactamase activity.
For BLIP-I, a few transconjugant colonies grew on Amp plates, indicating that inhibition was partially effective under the tested conditions.

**Learn**
Through this validation cycle, we learned the importance of experimental adaptability and quantitative refinement. Even without IPTG, the leaky expression of lacZ in BBa\_J435320 was sufficient to visualize conjugation events, confirming that our screening system was robust and reliable.

The partial inhibition observed in BLIP-I transconjugants suggests that BLIP activity may vary depending on expression levels or antibiotic concentration. If additional time were available, we would perform dose–response assays using graded ampicillin concentrations to better characterize the efficacy range of BLIP-I. These insights will inform the next design cycle and help optimize BLIP constructs for more consistent β-lactamase inhibition in the future.

## Wet Lab PCR Cycle 1/2

**Design**
We aimed to amplify the BLIP-I and BLIP-II gene sequences using PCR to generate sufficient DNA for cloning and downstream analysis. The goal was to obtain visible, high-quality DNA bands with sufficient concentration for ligation and digestion, while minimizing non-specific amplifications.

**Build**
To achieve this, we planned a 10 µL PCR reaction containing:
- 1 µL of BLIP template DNA (same for BLIP-I and BLIP-II)
- 0.5 µL each of VR and VF2 primers
- 3 µL of ddH2O
- 5 µL of 2x Taq Master Mix

We referenced the experimental handbook provided by our collaborating team, NYCU Formosa, and online resources to design our PCR program:
- Initial denaturation: 95 °C for 5 min
- 25 cycles of:
→ Denaturation: 95 °C for 30 seconds
→ Annealing: 55 °C for 30 seconds
→ Extension: 72 °C (1 min per kb + 30 seconds)
→ Final extension: 72 °C for 10 min
→ Hold: 4 °C
Based on sequence lengths including mCherry, we set the expected extension time to 1 minute 30 seconds for BLIP-I (1,609 bp) and 2 min 30 s for BLIP-II (1,982 bp).

**Test**
In the first trial, we observed strong and bright DNA bands at the expected size but also faint trailing smears behind the main band.
This suggested potential primer dimerization or underextension, likely caused by an insufficient extension time. According to the procedure, the extension step for BLIP-I should be 2 minutes; however, we failed to account for the additional length of the mCherry sequence in our construct, leading to a miscalculation of the required extension duration.

**Learn**
We concluded that short extension time contributed to the background products. We modified the extension to 2:00 for BLIP-I and 2:30 for BLIP-II, which yielded cleaner bands suitable for downstream cloning.

## Wet Lab PCR Cycle 2/2

**Design**
Use PCR to amplify BLIP-I and BLIP-II at sufficient yield and purity for downstream restriction digestion and cloning.

**Build**
Building on the previous cycle, where the PCR conditions were optimized, we aimed to further improve the yield and clarity of the BLIP-II amplification. After observing dim bands and low concentrations in several attempts, we modified the procedure while keeping the optimized temperature profile from DBTL cycle 1.

We maintained an initial denaturation at 95 °C for 5 minutes, followed by 35 total cycles (instead of 25) to increase DNA yield. Each cycle included denaturation at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds, and extension at 72 °C for 2 minutes (BLIP-I) or 2 minutes 30 seconds (BLIP-II), ending with a final extension at 72 °C for 10 minutes and hold at 4 °C. The total reaction volume was increased to 20 µL to compensate for some DNA loss during the gel extraction.

**Test**
Restriction digests on post-cleanup PCR products were more reliable.
Correcting the extension time removed most primer-dimers/smearing (trailing bands).
Bands were brighter than earlier attempts, though still not as intense as the first two runs.

**Learn**
The earlier smearing/dimerization in cycle 1 was driven by extension time mis-setting. With the corrected time setting, the specificity has greatly improved. The persistently dim bands/low concentration despite many scaling up and improvements suggest DNA template degradation due to repeated freeze-thaw.

Lesson learned: We should always aliquot templates to avoid repeated freeze-thaw process and subsequent template degradation.

Overall, the revised program produced clean, digest-ready amplicons, enabling downstream cloning with higher consistency.

## Wet Lab Gel Extraction Cycle 1/3

**Design**
To isolate pure fragments of BLIP-I, BLIP-II, and pSB1C3 using agarose gel electrophoresis while preventing too much DNA loss.

**Build**
We built the gel extraction protocol with the reference to the protocol from the NYCU lab manual. To purify DNA from the agarose gel, the desired bands are excised and transferred to labeled tubes. Add Buffer B after cutting the desired band on the gel, and incubate the mixture at 65 °C until the gel dissolves completely. The solution is transferred to a spin column, washed sequentially with Buffer W1 and Buffer W2, and centrifuged to remove impurities. The DNA is then eluted twice with 20 µL of ddH2O, and the purified DNA concentration is measured before storage at -20 °C.

**Test**
After gel extraction, we can only evaluate whether we got our desired bands and if they have high enough concentration after restriction digestion and running gel electrophoresis.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/e1.png)

09/23 Gel electrophoresis of BLIP-I, BLIP-II, and pSB1C3 restriction digestion

After several rounds of gel extraction and restriction digestion, the gels consistently showed faint or even non-visible DNA bands. We made multiple adjustments to reagent volumes and conditions, but none resulted in stronger bands. Eventually, after going over resources and the troubleshooting guide provided by GeneDireX, we discovered that Buffer W2 from the gel extraction kit had not been properly diluted with ethanol. This oversight likely caused inefficient DNA precipitation and binding to the silica column during the wash step, and therefore led to the low DNA recovery, as suggested by the weak or undetectable bands observed in our earlier attempts.

**Learn**
We invested a great deal of time and effort trying to identify the cause of our dim bands and low DNA concentrations. Overlooking the need to dilute Buffer W2 with ethanol significantly impacted our experimental progress, leaving us with limited time for subsequent adjustments and trials. This experience taught us an important lesson, always double-check the instructions for kits and reagents before starting any experiment to avoid losing valuable time over an easily preventable mistake.

## Wet Lab DBTL Gel Extraction Cycle 2/3

**Design**
Aims to isolate pure fragments of BLIP-I, BLIP-II, and pSB1C3 using agarose gel electrophoresis while preventing too much DNA loss.

**Build**
After figuring out we should dilute our Buffer W2 with ethanol beforehand, we used the same protocol from the NYCU experiment notebook.

**Test**
To our surprise, the DNA concentration remained low even after properly diluting Buffer W2 with ethanol. While this adjustment slightly improved the results, it became clear that another underlying factor was still causing the dim or non-visible bands observed after gel extraction. After researching and brainstorming over potential reasons, we suspected that pH level of ddH2O may be ideal, as the GeneDireX manual explicitly stated the ideal pH range to be 7.0-8.5. After measuring the pH of our ddH2O, we found that the pH was around 5-6, which could be the major reason for low elution strength. To ensure consistency, we modified the procedure by replacing ddH2O with Tris Buffer, as suggested by the manual.

**Learn**
Through this process, we learned that even small details in reagent choice can significantly affect DNA recovery efficiency. Replacing ddH2O with Tris buffer greatly improved the elution, as Tris helps maintain a stable pH that prevents DNA from adhering too tightly to the column membrane and protects it from acid-induced degradation. This experience emphasized the importance of understanding the chemical mechanisms behind each step rather than following protocols mechanically, allowing us to troubleshoot more effectively and improve the reliability of our gel extraction results.

## Wet Lab DBTL Gel Extraction Cycle 3/3

**Design**
Aims to isolate pure fragments of BLIP-I, BLIP-II, and pSB1C3 using agarose gel electrophoresis while preventing too much DNA loss.

**Build**
Following the protocol provided in the Gene DireX Inc. gel extraction kit, we purify DNA from the gel. 500 µL of Buffer B is added to the gel slice and incubated at 65 °C until fully dissolved. The solution is transferred to a spin column, washed with Buffer W1 and Buffer W2, and centrifuged to remove impurities. To make the DNA more concentrated, the DNA is eluted with 25 µL instead of 50µL of Tris Buffer (instead of ddH20 due to acidity), and stored at -20 °C.

**Test**

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/e2.png)

Restriction digestion results using the DNA samples purified by the modified protocol. The bands are visible and clean.

The purification went well and we purified our desired bands. After restriction digestion, we see bands indicating successful gel extraction. Proving that the acidic ddH2O is the reason for past failed attempts, and replacing it with Tris buffer works more effectively, we achieved clearer digested bands and substantially higher DNA yield.

**Learn**
We learned that the acidic ddH2O used during gel purification was the primary cause of low elution efficiency and reduced DNA concentration. By switching to Tris buffer for elution, we significantly improved DNA recovery and stability, ensuring sufficient yield for downstream reactions.

Using the purified PCR products from this optimized workflow, we proceeded with restriction digestion, ligation, and transformation, all of which were successful. This marked a major engineering milestone in our cloning workflow, demonstrating that our systematic optimization effectively resolved previous bottlenecks and enabled reliable plasmid construction.

## Wet Lab Making Competent Cell Cycle 1/2

**Design**
After ligation, our recombinant plasmid is ready to be introduced into the bacteria via transformation. To make our donor and recipient ready to take in the recombinant plasmid, we made competent cells for S17-1 and DH5α.

**Build**
Taking the protocol from the RBC HIT Competent Cell Protocol Book. To prepare competent E. coli cells, 0.6 mL of overnight culture was added to 12 mL of LB broth and incubated at 37 °C with shaking for about 1 hour until the OD600 reached 0.2-0.3. The culture was chilled on ice, centrifuged, and washed twice with ice-cold 10% CaCl2 to make the cells competent. Resuspend the final pellet in a small volume of 10% CaCl2 for immediate heat-shock transformation for the most effective approach, or in 15% glycerol + CaCl2 solution for long-term storage at –80 °C.

**Test**
After incubating the overnight culture, added to 12 mL of LB Broth, and incubate in 37 °C with shaking for 1 hour, we got OD600 = 0.275, which is within the desirable range of 0.2~0.3 OD. However, only a very tiny and almost non-visible pellet is observed after the first spin down. We further centrifuge it a few more times in order to get bigger pellets, but the pellets were still extremely small. As a result, the procedure was halted and we had to redo a new batch.

**Learn**
From the tiny pellets observed in our first competent cell preparation attempt, we realized the failure was likely due to low cell/DNA concentration. In addition, repeatedly centrifuging the culture may have further reduced cell potency. This experience taught us that both incubation timing and protocol accuracy are critical. To improve, we planned to incubate the overnight culture longer to achieve a higher OD and greater DNA concentration, and we also modified the procedure accordingly.

## Wet Lab Competent Cell Cycle 2/3

**Design**
Aimed to prepare E. coli S17-1 with higher OD values to ensure sufficient cell density and improve competency. By optimizing the preparation conditions, we intended to maximize transformation efficiency and establish both strains for use in later conjugation experiments.

**Build**
We took the protocol from the RBC HIT Competent Cell Protocol Book. To prepare competent E. coli cells, 0.6 mL of overnight culture was added to 12 mL of LB broth and incubated at 37 °C with shaking for about 3-4 hours until the OD600 reached 0.4-0.6. The culture was chilled on ice, centrifuged, and washed twice with ice-cold 10% CaCl2 to make the cells competent. Resuspend the final pellet in a small volume of 10% CaCl2 for immediate heat-shock transformation for the most effective approach, or in 15% glycerol + CaCl2 solution for long-term storage at -80 °C.

**Test**
After incubating the overnight culture for more than 4 hours, we measured an OD600 of 0.8, indicating high cell density. Unlike our first attempt, a visible pellet was obtained after the initial spin down, showing clear improvement. However, we could not proceed directly to heat-shock transformation since no plasmid was yet fully prepared for transformation. As a result, we stored 10 tubes as glycerol stocks at –80 °C. Unfortunately, after being stored for several weeks, the S17-1 glycerol stocks lost competency, and subsequent transformation attempts were unsuccessful.

**Learn**
From the loss of competency in our glycerol stock, we realized that S17-1 cells are highly sensitive to long-term storage and quickly lose their ability to take up DNA. This taught us the importance of using freshly prepared competent cells rather than relying on frozen stocks for transformations. We also learned that timing the preparation of competent cells to align with plasmid availability is critical to avoid unnecessary storage and reduced efficiency.

## Wet Lab Competent Cycle 3/3

**Design**
For our third cycle, we aimed to prepare E. coli S17-1 cells with a high OD value and ensure they were freshly made chemically competent. Unlike our previous attempts, where glycerol stocks lost competency or the pellets were too small, this time we planned to use the cells immediately after preparation. This approach was designed to maximize the likelihood that the donor strain would successfully take up the plasmid.

**Build**
We followed the same protocol as in our second cycle, but with one key change: instead of making glycerol stock of the competent cells, we used them immediately after preparation for transformation.

**Test**
After incubating the overnight culture for 4 hours, we measured an OD600 of 0.392 (~0.4), and a clear, visible pellet was obtained after the initial spin down. We then directly performed heat-shock transformation using the plasmid we had miniprepped earlier. Following the transformation, clear colonies were observed, confirming that our approach with freshly made competent cells was successful.

**Learn**
We learned that the freshness of competent cells is crucial for transformation success. Using freshly prepared S17-1 cells significantly improved our results, highlighting how subtle differences in preparation can directly determine experimental outcomes.

## Dry Lab Cycle Conjugation 1/2

**Design**
The initial design of our conjugation model was to input quantitative data from wet lab experiments directly into our mathematical equations framework in order to produce realistic simulations and visualizations of bacterial conjugation.

**Build**
To model the dynamics of donors, recipients, and transconjugants, we applied mathematical formulas that describe recipient and transconjugants in an inversely proportional manner. These formulas capture the essential behavior of conjugation: as transconjugants increase, recipients decrease correspondingly, while donors provide a stable source of plasmids.

**Test**
During finalization, we found that assuming donors remain constant did not fully capture real-life dynamics, as donors actually grow over time. Additionally, due to temporal constraints in the experimental flow, the wet lab was unable to provide the necessary data within our modeling timeline.

**Learn**
Although real-life experimental data from the wet lab were unavailable within the project timeline, our dry lab team took the initiative to research existing literature and derive our own parameter values. This approach allowed us to refine our conjugation model despite the lack of direct measurements.
Moreover, computational modeling enabled us to simulate scenarios that the wet lab could not feasibly achieve, providing a broader platform for exploring plasmid transfer dynamics.

## Dry Lab Cycle Conjugation 2/2

**Design**
We developed a conjugation model that simulates both donor growth and no-donor-growth scenarios. The no-donor-growth version provides a clear view of the inverse relationship between transconjugants and recipients, isolating the direct effect of conjugation without the added complexity of donor proliferation. Because we operate as a dry lab, we are able to construct and analyze this simplified model—something that would be difficult or impossible to replicate in the wet lab—thereby offering unique insights into plasmid transfer dynamics under controlled conditions.

**Build**
We proceeded to construct the model using parameter values derived from accepted experimental ranges: donor and recipient populations of 1.0×106 and 1.0×107 cells/mL, respectively, reflecting the common 1:10 ratio used in conjugation experiments. This enables us to maintain progress despite the absence of primary data.

**Test**
The values were implemented into our equations to model how the donor, recipient, and transconjugant populations change over time. This helped us ensure that our model behaves in a relatively realistic way and makes biological sense under the assumptions we set. After inputting these values, we generated graphs to provide clearer visualization of the population dynamics and make the results more intuitive to interpret.

**Learn**
Simulation results show that in the no-growth model, donors remain constant, serving as a stable plasmid source and making the inverse relationship between recipients and transconjugants clearly visible. When donor and recipient growth are included, cell numbers increase exponentially, amplifying plasmid transfer due to the expanding donor population. By comparing these two scenarios, our dry lab simulation achieved something the wet lab cannot easily replicate—modeling and visualizing plasmid transfer under controlled, hypothetical conditions—highlighting both the direct impact of conjugation and the amplification effect of cell growth on resistance gene spread.

## Dry Lab Cycle - BLIP Expression

**Design**
We simulate a model in COPASI to explore the interaction between BLA and its inhibitors-BLIP-I and BLIP-II. This design step defines the key parameters and reaction networks needed to compare how each BLIP variant affects Beta-Lactamase (BLA) expression and activity.

**Build**
We build the model by implementing the defined reactions and expression profiles of BLA, Ampicillin (Amp), BLIP-I, and BLIP-II into COPASI. This includes specifying rate laws, initial concentrations, and any relevant interactions to ensure that the model represents the biological system.

**Test**
We test the model by running multiple simulations to evaluate BLA dynamics under the influence of each BLIP variant. These simulations generate time-course data, allowing us to compare the inhibitory efficiency of BLIP-I and BLIP-II in modulating BLA activity.

**Learn**
We learn from the analysis of the simulation outputs and graphs, which show that BLIP-II achieves more effective inhibition of BLA than BLIP-I. These insights inform future model refinements and experimental strategies for validating BLIP-mediated control of BLA.

## HP DBTL Cycle Feedback and Interview

**Design**
At the beginning of our Human Practices work, our goal was to gather expert insights on antibiotic resistance, gene transfer safety, and the ethical implications of conjugation-based systems. We designed interview questions intended to guide our project direction and validate our ideas. However, our early drafts were too broad or unrelated, making it difficult for interviewees to provide specific or actionable feedback.

**Build**
We conducted several initial interviews with professors and field experts, using the original question sets. During these sessions, we realized that some questions lacked focus or were too general to yield meaningful responses. To improve, we began collaborating internally across subteams, especially between the Wet Lab, Dry Lab, and HP members. This was to ensure that future interview questions were directly connected to our project's experimental design and goals.

We also established a post-interview reflection process, where team members would discuss what worked well and what could be improved for future stakeholder engagements. In addition to that, we wanted to see our impact in our outreach community, thus we held interviews with in-school students and the head principal.

**Test**
In subsequent interviews, the revised question sets produced more insightful and relevant feedback. Interviewees were able to engage in deeper discussion about our project's biosafety considerations, educational value, and real-world applications. These focused interviews provided information that directly informed our project implementation and wiki documentation.

The addition of post-meeting team discussions helped us identify key takeaways more clearly and brainstorm how each piece of feedback could be integrated into our design and outreach strategy.

**Learn**
We learned that Human Practices requires the same iterative refinement as wet-lab experiments — careful planning, testing, and reflection lead to better outcomes.
Through this cycle, we discovered that:
- Collaborative question design with relevant subteams produces more meaningful and technically grounded discussions.
- Focused, specific questions yield deeper expert insights compared to general or abstract prompts.
- Conducting post-interview debriefs ensures that valuable feedback is not only documented but also actively integrated into project development.
This iteration marked a significant improvement in how we approach stakeholder engagement, transforming our interviews from simple Q&A sessions into productive dialogues that shaped our project's ethical and scientific direction.

## HP DBTL Cycle: 2-Day Education Program

**Design**
In the initial phase, our objective was to investigate the relationship between early childhood creativity and their understanding of antimicrobial resistance (AMR). We hypothesized that introducing AMR through interactive storytelling and games would allow young children to better grasp hygiene practices and the dangers of antibiotic misuse. This was grounded in the idea that instilling basic awareness at an early age could foster responsible habits and future scientific curiosity.

**Build**
In order to construct this outreach initiative, we designed a series of hands-on learning stations tailored to children of ages 4~5. This included reading a hand-illustrated storybook titled, “Lil Joey and the Jungle’s Sick Story” which introduced the concept of AMR and how we can prevent them. Other activities that promoted young creativity in the field of synthetic biology include decorative games like “Build-A-Bug-Biolab” that allowed kids to showcase their interests in editing their own bacterias to make changes to the fields of biology. In addition to that, our team prepared materials that can be further used in the future for children of all ages, this includes our coloring book, educational videos, and more. This program was crafted to appeal to children’s senses of imagination while simplifying scientific principles for their age level.

**Test**
The two-day program was implemented at Star Academy in Hsinchu, successfully engaging over 130 kindergarten students across multiple rotating activity stations. The children demonstrated active participation during storytelling sessions by frequently raising their hands to ask questions and volunteering their ideas on preventing the spread of germs and infections. They also made insightful comments about medication misuse when prompted during the story of “Lil Joey and the Jungle’s Sick Story”. In the Build-A-Bug Biolab station, students creatively designed “problem-solving bacteria,” reflecting their ability to grasp abstract biological concepts through play. Many children drew bacteria with “superpowers” like fighting sickness, eating bad germs, or healing wounds, early evidence of their developing understanding of microbial functions. Teachers and staff noted the high level of excitement and retention, and several expressed interest in incorporating similar modules into their own teaching. This strong engagement validated our approach and confirmed that early childhood education can lay foundational knowledge for topics like AMR and responsible health behavior.

**Learn**
After our outreach at Star Academy, we realized that for younger audiences, interactive movement-based learning and simplified analogies significantly improved engagement and understanding. While our original plan already included visuals and stories, seeing how quickly students responded to physical games like the “No Germs Alive” activity helped us shape our future outreach planning by encouraging us to incorporate more interactive elements and clear debrief moments, even for older age groups, to ensure the understanding of AMR concepts in both the medical and environmental field through multi-sensory learning.

## HP DBTL Cycle : Public Hospital Booth

**Design**
In this cycle, our objective was to explore the relationship between community awareness and attitudes toward antibiotic use in vulnerable populations. We hypothesized that elderly patients, with proper guidance, would adopt more responsible behaviors to reduce AMR risks. This was grounded in the belief that increasing public literacy could reduce misconceptions and improve health outcomes.

**Build**
Firstly, we identified the primary aim of our outreach and constructed our plan around it. We carefully designed brochures that clearly explained key elements: the beginning of our journey and our goals, the purpose of our outreach, the impact and harm of the issue we are addressing, and our previous collaboration with Ton Yen Hospital. Once the brochures were completed, our Human Practices team carefully planned the outreach schedule and determined how best to approach the audience. To ensure the effectiveness of the outreach, we collaborated with Ton Yen Hospital once again and developed detailed interactive questions about the dangers of AMR. In addition, we prepared small rewards, provided through our collaboration with the Hsinchu Health Bureau, for participants who answered the questions and demonstrated their support.

**Test**
To assess the effectiveness of our outreach strategy at Ton Yen Hospital, we directly engaged with the local elderly population. During our information booth sessions, we distributed brochures that contained information about the project and constructed interactive Q&A sessions. The questions, which were based on common misconceptions about antibiotic use allowed the elderly community to actively participate and interact with the team. We asked a total of 5 questions where people voluntarily raised their hands to answer questions.

**Learn**
Through this activity, we learned that antimicrobial resistance still remains unfamiliar to older age groups, with many people having misconceptions regarding prescription and taking antibiotics. We also acquired the information that iGEM isn’t as well known for Taiwanese citizens, and the synthetic biology field still remains more foreign.

## HP DBTL Cycle: Guiding Future Graduates

**Design**
In this cycle, our objective was to examine the relationship between student career aspirations and their engagement with AMR education. We hypothesized that linking AMR prevention to students’ future goals in medicine or biology would increase both interest and responsibility toward scientific issues. This was based on the notion that contextualizing synthetic biology within real career pathways enhances relevance and motivation.

**Build**
We initiated the process by designing a lesson plan to map out the slideshow we'd use during the showcase. The presentation included an opening question, relations to the SDGs, brief overview of the issue of antimicrobial resistance, our team, interactive questions, and an exit ticket. Our questions were mainly based on the feedback we’ve acquired from previous interviews and past teams’ works. Not only do we intend to observe the relationship between student career aspirations with AMR education, it is also meant to align with the Sustainable Development Goals (SDGs) to show how our project shows mindful connection.

**Test**
To assess the impact and actual implementation of our experiment, we consulted other advisors beforehand about whether they think the lesson will be comprehensible for students. We also asked if there could be connections to broader and more complex topics, as well as the implementation of our event into the curriculum.

**Learn**
They advised us to include more visuals such as videos to draw people's interest, enhance comprehension, and keep them attentive, which we then used to amend our slides accordingly. From this event, we learned that for our outreach purposes many students got inspired by our events and have an interest in pursuing a career in similar fields.

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![Top Image](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/lightbluetitlebg.png)

# Experiments

LIGATION
RESTRICTION ENZYME MAPPING
POLYMERASE CHAIN REACTION (PCR)
GEL EXTRACTION(8/26 - 9/20)
GEL EXTRACTION(SINCE 9/26)
PREPARATION OF LB AGAR AND LB BROTH
PREPARATION OF LB AGAR/BROTH WITH ANTIBIOTIC
ISOLATION OF A SINGLE COLONY
REHYDRATION
GLYCEROL STOCK
REVIVING GLYCEROL STOCK
REPARATION OF CHEMICALLY COMPETENT CELLS
HEAT SHOCK TRANSFORMATION
EXTRACTION OF PLASMID DNA (MINIPREP)
RESUSPENSION OF THE SAMPLE FROM THE DISTRIBUTION KIT
X-GAL
CONJUGATION
REFERENCES

### LIGATION

Objective - To join the restriction-digested pSB1C3 vector and BLIP insert into a single circular plasmid using T4 DNA ligase for downstream transformation and validation.

**Materials & Equipment**

- Restriction-digested pSB1C3 (vector)
- Restriction-digested BLIP fragment (insert)
- 10x T4 DNA ligase buffer
- ddH2O (nuclease-free)
- T4 DNA ligase (keep on ice)
- 0.2 mL PCR/ligation tubes, tube rack
- Microcentrifuge (brief spin)
- Ice bucket / cold block
- Incubator or fridge set to 4 °C
- Competent cells for transformation (e.g., S17-1 or DH5α)
- Pipettes and sterile tips

**Protocol**

1. Label a sterile 0.2 mL tube with the sample ID and date.
2. Add 2.0 µL of digested pSB1C3 (vector).
3. Add 2.0 µL digested BLIP (insert).
4. Add 2.0 µL of 10x T4 DNA ligase buffer.
5. Add ddH2O to bring the total volume to 19.5 µL.
6. Gently mix the mixture by pipetting up and down or flicking, then briefly spin it to collect liquid.
7. Add 0.5 µL of T4 DNA ligase (the enzyme was kept on ice prior to adding).
8. Briefly spin the tube and mix gently.
9. Incubate the reaction at 4 °C for 12-16 hours (overnight), and store the tube rack in the fridge during incubation.
10. After incubation, 10 µL of the ligation mix can be use for transformation. Store the remaining ~10 µL at -20 °C. [17]

Note: If ligation with 2 µL vector + 2 µL insert failed, the insert volume was increased while keeping the total volume ≤ 20 µL. Common insert: vector ratios included **3:1, 5:1, and 7:1**.

**Troubleshooting & Failures**

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| Insert failed to ligate | The inserted DNA concentration is too low | Increase the insert volume while keeping the final reaction volume constant. Test different insert:vector molar ratios (commonly 3:1, 5:1, or 7:1). Alternatively, concentrate the digested insert before ligation. |
| Backbone re-ligated without insert (self-ligation) | Purify the digested vector and insert fragments by gel extraction before ligation. This removes uncut plasmid and small digestion byproducts. |

### RESTRICTION ENZYME MAPPING

Objective - To verify plasmid identity by digesting purified plasmid DNA with specific restriction enzymes and analyzing the resulting fragments via agarose gel electrophoresis to generate a simple restriction map.

**Materials & Equipment**

I. Restriction Digestion
- Purified plasmid DNA
- Restriction enzyme: EcoRI, PstI
- 10x CutSmart Buffer
- Sterile ddH2O
- Ice box

II. Agarose Gel Electrophoresis
- Agarose powder
- 1x TAE buffer
- DNA ladder
- 6x loading dye
- SYBR Safe (for staining)
- Gel casting tray and electrophoresis chamber
- UV transilluminator

Important Notes on Enzyme Use:
- Always add restriction enzymes last and keep them on ice at all times.
- Use a new pipette tip for each enzyme to avoid contamination.
- Since enzyme stocks contained 50% glycerol, use less than 1/10 of the total reaction volume to prevent inhibition.

**Protocol**

I. Restriction Digestion

1. Add 5 µL of plasmid DNA (~500 ng)to a labeled microcentrifuge tube.
2. Add 2 µL of 10x CutSmart Buffer.
3. Add sterile water to bring the total volume to 20 µL. Gently mix the mixture was tapping or pipetting and briefly spin down.
4. Add 0.5 µL (~5 units) of each restriction enzyme. Gently mix the mixture and spin down briefly again.
5. Incubate the reaction at 37 °C for 1 hour, then heat-inactivate at 80 °C for 20 minutes.
6. Store the reaction product at -20 °C until use.
7. Perform gel electrophoresis to confirm the digestion products.
Note: According to NEB, 1 unit of enzyme was defined as the amount required to digest 1 µg of λ DNA in a 50 µL reaction at 37 °C for 1 hour.

II. Agarose Gel Electrophoresis

1. Weigh the 0.5 g of agarose powder into a beaker.
2. Add 50 mL of 1x TAE buffer and mix gently.
3. Heat the solution on a hot plate until the agarose dissolves completely (the solution becomes clear).
4. Allow the solution to cool slightly (~60 °C).
5. Add 5 µL of SYBR Safe dye.
6. Pour the solution into a casting tray with a comb, and remove any bubbles.
7. Leave the gel to solidify at room temperature (~20-30 minutes).
8. Carefully removed the comb, leaving the gel ready for electrophoresis.
9. During sample preparation, add loading dye to the DNA samples with a dye ratio of 5:1.
10. Run the gel at 100 V for approximately 30 minutes, or until the bromophenol blue dye reaches the second-to-last line (around 600 bp in 1% agarose).
11. Visualize the DNA bands using a UV transilluminator. Capture the images, and attach the printout to the lab notebook.[17]

**Troubleshooting & Failures**

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| The product is not digested | Incubation time is too short | Ensure sufficient incubation (typically 1-3 hours, or as recommended by the enzyme supplier). Preheat the incubator, or use a calibrated heat block. |
| The enzyme is not mixed evenly into the reaction | Gently mix the mixture up and down before incubation. Avoid vigorous vortexing. |
| Low enzyme activity (loss of function) | Keep enzymes on ice at all times during setup and promptly return to -20 °C after use. |
| Reaction is inhibited due to excess glycerol | Since enzyme stocks contain 50% glycerol, the total glycerol should not exceed 10% of the final reaction volume (≤ 1/10 of total volume). |
| Low concentration of the digested product | DNA degradation | Keep samples on ice during setup, and store final products at 4 °C. |
| The starting concentration of the PCR product is too low | Consult the **PCR Troubleshooting (Low Concentration Product)** guidelines to improve DNA yield before digestion. |

### POLYMERASE CHAIN REACTION (PCR)

Objective - To amplify the BLIP-I and BLIP-II genes to verify successful transformation. This procedure generates sufficient DNA for downstream verification and analysis.

**Materials & Equipment**

- PCR machine
- VR primer
- VF2 primer
- Resuspended BLIP template DNA
- ddH2O
- 2x Taq Mastermix

**PCR cycling condition settings:**

- Initial denaturation: 95 °C for 5 minutes.
- 35 cycles of:
→ Denaturation: 95 °C for 30 seconds.
→ Annealing: 55 °C for 30 seconds.
→ Extension: 72 °C for (1 minute per kb + 30 sec)—-2 minutes for BLIP-I and 2 minutes 30 sec for BLIP-II.
- Final extension: 72 °C for 10 minutes.
- Hold: 4 °C
- BLIP-I & BLIP-II concentration: 10 ng per reaction (typical range: 10-50 ng).

**Protocol**

1. Add 0.5 µL of VF2 primer and 0.5 µL of VR primer to a sterile PCR tube.
2. Add 1 µL of BLIP template DNA.
3. Add 3 µL of ddH2O.
4. Add 5 µL of 2x Taq Mastermix (Add reagents from least to most concentrated).
5. Store the mixture temporarily at **4 °C**. [17]

Notes: Remember to pipette mix the solution and mix the restriction enzymes before use.

**Troubleshooting & Failures**

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| Low concentration of products | The number of PCR cycles was insufficient | The number of PCR cycles was increased, while avoiding overcycling that could cause non-specific amplification. |
| The input DNA quality or quantity was too low | Check the DNA quality, and increase the input amount if necessary. Confirm the template is not degraded. |
| Dimerization (two products or unintended bands ligating together) | Non-optimal PCR conditions (e.g., too high primer concentration, low annealing temperature, or overly long primers) | Optimize the PCR conditions by lowering primer concentration, increasing annealing temperature, and verifying primer design. Use high-quality templates and reagents to minimize contaminants. |
| Smeared product | Too many PCR cycles | Optimize PCR conditions to reduce cycle numbers and prevent product smearing. |
| No band | Low concentration of template, primer concentration too low, or annealing temperature too high | Check template presence, reduce annealing temperature, or verify gel electrophoresis setup. |

### GEL EXTRACTION (INITIAL PROCEDURE, USED FROM 8/26 TO 9/20)

Objective - To isolate pure fragments of BLIP-I, BLIP-II, and pSB1C3 using agarose gel electrophoresis.

**Materials & Equipment**

- DNA electrophoresis materials
- Gel-purified plasmids or ligation products
- Spin column
- Heat block
- Buffer B (dissolves gel)
- Buffer W1 & W2 (ethanol wash)
- ddH2O (pH 7.0)

**Protocol**

Agarose Gel Electrophoresis

1. Weigh the 0.5 g of agarose powder into a beaker.
2. Add 50 mL of 1x TAE buffer and mix gently.
3. Heat the solution on a hot plate until the agarose dissolved completely (the solution becomes clear).
4. Allow the solution to cool slightly (~60 °C).
5. Add 5 µL of SYBR Safe dye.
6. Pour the solution into a casting tray with a comb, and remove any bubbles.
7. Leave the gel to solidify at room temperature (~20-30 minutes).
8. Carefully remove the comb, leaving the gel ready for electrophoresis.
9. During sample preparation, add loading dye to the DNA samples with a dye ratio of 5:1.
10. Run the gel at 100 V for approximately 30 minutes, or until the bromophenol blue dye reaches the second-to-last line (around 600 bp in 1% agarose).
11. Visualize the DNA bands using a UV transilluminator. Capture the images, and attach the printout to the lab notebook.
12. Excise desired bands using a clean (DNA-free) scalpel.

Gel Extraction

13. Add 500 µL of Buffer B to the gel slice, and incubate the mixture at 65 °C until the gel fully dissolves (~10 minutes, swirling every 2 minutes). Cool the solution to room temperature.
14. Transfer the dissolved gel solution to a spin column and centrifuge at 14,000 rpm for 1 minute. Discard flow-through.
15. Add 400 µL of Buffer W1, centrifuge for 1 minute. Discard flow-through.
16. Add 600 µL of Buffer W2 and spin for 1 minute. Discard flow-through.
17. Spin again at 14,000 rpm for 2 minutes, then open and air-dry for 2 minutes to remove residual ethanol.
18. Place the column into a new labeled tube. Add 25 µL of ddH2O directly onto the membrane. Incubate the tube for 2 minutes at room temperature, then spin at 14,000 rpm for 2 minutes. Collect the supernatant, reapply to the membrane, and spin again for a second elution.
19. Measure DNA concentration (OD260/280), and store the purified DNA at -20 °C.[17]

**Troubleshooting & Failures**

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| Low concentration of purified DNA | The gel did not fully melt | Incubate the sample longer at 65 °C and gently mix (vortex or rotate) until the gel is completely dissolved before loading. |
| The elution volume is too large | Reduce the elution volume. If the concentration remains low, perform an additional wash with Buffer W2, followed by an extra dry spin. |
| The column is not completely dry | After the final wash, centrifuge the sample for 2-3 minutes and briefly air-dry before elution. |
| Excessive UV exposure during band cutting damaged the DNA | Minimize UV exposure by turning off the blue-light transilluminator immediately after capturing the image, and use the saved image for analysis instead of repeatedly exposing the gel to the light. |

### GEL EXTRACTION (PROCEDURE PROVIDED FROM THE GEL EXTRACTION KIT, USED SINCE 9/26)

Objective - To isolate pure fragments of BLIP-I, BLIP-II, and pSB1C3 while minimizing DNA loss.

**Materials & Equipment**

- DNA electrophoresis materials
- Gel-purified plasmids or ligation products
- Spin column
- Heat block
- Buffer B (dissolves gel)
- Buffer W1 & W2 (ethanol wash)
- BE buffer (pH 7.0)

**Protocol**

Agarose Gel Electrophoresis

1. Weigh 0.5 g of agarose powder into a beaker.
2. Add 50 mL of 1x TAE buffer and mix gently.
3. Heat the mixture on a hot plate until the agarose dissolves completely (the solution becomes clear).
4. Allow the solution to cool slightly (~60 °C).
5. Add 5 µL of SYBR dye.
6. Pour the solution into a casting tray with a comb, and remove the bubbles if present.
7. Leave the gel to solidify at room temperature (~20-30 minutes).
8. Remove the comb carefully, and the gel is ready for electrophoresis.
9. During sample preparation, add loading dye to the DNA samples at a dye-to-sample ratio of 5:1.
10. Run the gel at 100 V for ~30 minutes, or until the bromophenol blue dye reaches the second-to-last line (~600 bp in 1% agarose).
11. Visualize DNA bands using a UV transilluminator, and capture and attach the images to the lab notebook.
12. Excise the desired DNA fragments from the agarose gel.
13. Transfer each gel slice (up to 300 mg) to a 1.5 mL microcentrifuge tube.
14. Add 500 µL of Buffer B to the sample, mix by vortexing, and incubate at 60 °C for 10 minutes (or until the gel slice is completely dissolved).
15. During incubation, the tube was mixed by vortexing every 2-3 minutes. The dissolved sample mixture was cooled to room temperature afterward.
16. Place a PG column into a collection tube. Apply the supernatant from step 15 to the PG column by decanting or pipetting.
17. Centrifuge the sample at 14,000 x g for 30 seconds.
18. Discard the flow-through, and place the PG column back into the same collection tube. *(If the sample exceeded 800 µL, repeat the DNA binding step.)*
19. Add 400 µL of Buffer W1 to the PG column and centrifuge at 14,000 x g for 30 seconds. Discard the flow-through, and place the column back into the same tube.
20. Add 600 µL of Buffer W2 (with ethanol added) to the PG column and centrifuge at 14,000 x g for 30 seconds. Discard the flow-through, and return the column to the same tube.
21. Centrifuge the column again at 14,000 x g for 2 minutes to remove residual Buffer W2.
22. Elute the DNA by placing the PG column into a clean 1.5 mL microcentrifuge tube.
23. Add 25 µL of Buffer BE (pH between 7.0 and 8.5) directly to the center of each PG column. Leave the sample to stand for 2 minutes, then centrifuge at 14,000 x g for 2 minutes. Repeat this elution step once. [6]

**Troubleshooting & Failures**

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| Low concentration of purified DNA | The gel did not melt completely | Incubate the sample longer at 65 °C and gently mix (vortex or rotate) until the gel is fully dissolved before loading. |
| The elution volume is too large | Reduce the elution volume. If the concentration remained low, perform a repeat wash with Buffer W2, followed by an additional dry spin. |
| The column was not completely dry | After the final wash, centrifuge the sample for 2-3 minutes and air-dry briefly before elution. |
| Excessive UV exposure during band cutting damaged the DNA | Minimize UV exposure by turning off the blue-light transilluminator immediately after capturing the image, and use the saved image for analysis instead of exposing the gel repeatedly. |
| Excess water diluted the DNA | Reload the elute onto the same column and centrifuge it again to recover any remaining DNA. |
| Buffer W1 not diluted | Dilute buffer W1 using ethanol. [6] |

### PREPARATION OF LB AGAR AND LB BROTH

Objective - To prepare sterile LB agar plates and LB broth as general media for bacterial growth. These plates are used in future bacterial isolation and culture.

**Materials & Equipment**

- LB agar powder
- LB broth powder
- Distilled water (ddH2O)
- Aluminum foil
- Autoclave tape
- Autoclave
- Stirring equipment (magnetic stirrer or heat and stir bar)
- Petri dishes
- Sterile centrifuge tubes or culture tubes
- Sterile bottles or flasks

**Protocol**

I. Prepared LB Agar (solid medium)

1. Mix 8 g LB agar powder with 250 mL ddH2O in a suitable container (32 g LB Agar Powder: 1L ddH2O).
2. Heat and stir until the powder is completely dissolved.
3. Cover the bottle opening with aluminum foil and secure it with autoclave tape (do not fully tighten the cap).
4. Sterilize by autoclaving at 121 °C for 30 minutes using moist heat.
5. Allow the LB agar to cool to approximately 65 °C, warm to the touch but not hot.
6. Pour the cooled agar into sterile Petri dishes, taking care not to let it solidify.

II. Prepared LB Broth (liquid medium)

1. Mix 6.25 g LB broth powder with 250 mL ddH2O (25 g LB Broth Powder:1 L ddH2O).
2. Heat and stir until completely dissolved.
3. Cover the bottle opening with aluminum foil and secure it with autoclave tape (do not fully tighten the cap).
4. Sterilize by autoclaving at 121 °C for 30 minutes using moist heat.
5. Aliquot the sterile LB broth into sterile centrifuge tubes or culture tubes (e.g., 10 mL or 5 mL portions).
6. Store at 4 °C until use (do not heat after sterilization). [17]

### PREPARATION OF LB AGAR/BROTH WITH ANTIBIOTIC

Objective - To prepare selective media containing antibiotics for screening and maintaining bacteria carrying resistance genes. This way, we are able to verify if conjugation is successful.

**Materials & Equipment**

- LB agar or LB broth (prepared as in “Preparation of LB Agar and LB Broth,” Steps 1-4)
- Appropriate antibiotic stock solution
- Sterile Petri dishes or culture tubes
- Pipettes and sterile tips
- Stirring equipment (for agar)

**Protocol**

1. Prepare LB agar or broth following the protocol in “Preparation of LB Agar and LB Broth” (Steps 1-4).
2. Allow the medium to cool to a temperature suitable for adding antibiotics (~45-55 °C).
3. Add the antibiotic at a 1:1000 dilution (Antibiotic: LB = 1:1000). Mix gently to avoid bubbles.
4. Pour agar into sterile Petri dishes or aliquot broth into sterile tubes.
5. Allow the agar to solidify or store the broth at 4 °C until use.

**Working Concentration for Each Antibiotic [17]**

| Chemical | Stock Concentration | Working Concentration |
| --- | --- | --- |
| Ampicillin | 50 mg/mL | 50 µg/mL |
| Chloramphenicol | 25 mg/mL | 25 µg/mL |
| Kanamycin | 50 mg/mL | 50 µg/mL |
| Tetracycline | 15 µg/mL | 15 µg/mL |
| IPTG | 0.1 M | 0.1 mM |
| X-gal | 20 mg/mL | 20 µg/mL |

### ISOLATION OF A SINGLE COLONY

Objective - To obtain well-isolated single bacterial colonies from a mixed culture. This ensures the establishment of clonal populations that can be used reliably in downstream experiments and characterization.

**Materials & Equipment**

- LB agar plates
- Bacterial suspension (from glycerol stock, broth culture, etc.)
- Bunsen burner (or alcohol lamp)
- Parafilm
- Permanent marker (for labeling)
- Incubator (set to 37 °C)
- (Quadrant Streak Method) Inoculating loop (reusable) or sterile disposable toothpicks
- (Spreading Method) Glass spreader (hockey stick)
- (Spreading Method) 95% ethanol (for spreader sterilization)
- (Spreading Method) Micropipette and sterile tips (for adding suspension, e.g., 100 µL)

**Protocol**

I. Quadrant Streak Method

1. Label the bottom of an LB agar plate.
2. Sterilize an inoculating loop by flaming it over a Bunsen burner until red hot and allow it to cool before use, or use a sterile disposable toothpick.
3. Dip into the bacterial suspension and streak a small section of the plate (first quadrant).
4. Sterilize the loop again, streak from the first quadrant into the second quadrant, spreading fewer cells.
5. Repeat for the third and fourth quadrants to obtain isolated colonies.
6. Incubate at 37 °C for 12-16 hours.
7. Pick well-isolated colonies for further experiments or seal the plate with parafilm and store it at 4 °C for further use.

II. Spreading Method

1. Label the bottom of the agar plate with bacterial host, treatment, antibiotic resistance, your name, and date.
2. Sterilize the glass spreader (hockey stick) as follows:
→ Immerse the spreader in 95% ethanol.
→ Remove it and allow excess ethanol to drip off.
→ Pass the spreader briefly through a flame to sterilize.
→ Allow it to cool before using it to spread the bacterial suspension.
3. Drop a suitable volume of bacterial suspension onto the plate (commonly 100 µL).
4. Use the cooled spreader to evenly spread the suspension across the agar surface until no visible liquid remains.
5. Allow the bacterial suspension to dry briefly on the plate.
6. Incubate the plate at an appropriate temperature (usually 37 °C) for 12-16 hours.
7. Pick well-isolated colonies for further experiments or seal the plate with parafilm and store it at 4 °C for further use.

**Troubleshooting & Failures**

I. Quadrant Streak Method

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| No single colonies | The inoculating loop is not sterilized between quadrants. | Flame-sterilize the loop after streaking each quadrant. [2] |
| No colonies at all | The loop is too hot after sterilization, killing the bacteria. | Allow the loop to cool for 5-10 seconds before touching bacteria, or first touch the agar to check cooling. [15] |
| Smearing, not isolated colonies | The loop is too hot after sterilization, killing the bacteria. | Gently streak across the agar surface without applying pressure. |
| No single colonies, only dense growth | The bacterial suspension is too concentrated. | Dilute the bacterial suspension before streaking. |

II. Spreading Method

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| No single colonies, lawn growth | Too many bacteria are plated, no dilution is performed. | Perform serial dilutions (10-1, 10-2, 10-3) and plate each dilution. [15] |
| No growth at all | The spreader is not cooled after flaming, killing the bacteria, or incorrect antibiotic selection. | Allow the spreader to cool before use; confirm the correct antibiotic in the plate. [10] |
| Contamination (unexpected colonies) | The spreader is not sterilized properly, or the plate is exposed to open air for too long. | Flame-sterilize the spreader each time; minimize plate exposure during spreading. [12] |
| Uneven growth (bacteria only in one area) | The suspension is not spread evenly, or the volume is too high. | Use a correct inoculum volume (≈100 µL) and evenly spread until no liquid remains. |

### REHYDRATION

Objective - To revive lyophilized E. coli S17-1 cells and establish an active bacterial culture. Since the cells are freeze-dried for shipping, proper rehydration and resuspension are required prior to use.

**Materials & Equipment**

- Sterile water
- Sterile broth (0.5 mL - 10 mL)
- LB agar plates (2)
- Bacterial sample
- Heat source (e.g., Bunsen burner or heating block)
- Sterile forceps/tweezers
- Micropipette and sterile pipette tips
- Sterile L-shaped spreader (hockey stick spreader)
- Shaker incubator (37 °C)
- Standard incubator (37 °C)

**Protocol**

1. Heat the outer tube at the tip (do not expose the bacterial pellet to heat).
2. Place a drop of sterile water on the heated area (thermal expansion helps loosen the outer tube).
3. Use sterile forceps to crack the outer tube tip carefully.
4. Gently remove the inner tube.
5. Remove the cotton plug.
6. Add 0.3-0.5 mL sterile broth into the inner tube to resuspend the bacterial pellet into a suspension.
7. Prepare 1 tube of 5-10 mL LB broth and 2 LB agar plates.
8. Pipette 0.1 mL of the bacterial suspension onto each LB agar plate.
9. Spread evenly using a sterile L-shaped spreader (hockey stick spreader).
10. Transfer the remaining suspension into the 5-10 mL LB broth tube.
11. Incubate:
- LB agar plates: 16 hours at 37 °C.
- LB broth: 16 hours at 37 °C with shaking at 200 rpm. [27]

**Troubleshooting & Failures**

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| No bacterial growth observed | Heat applied too close to the bacterial pellet, resulting in cell death. | Heat only the outer glass tip, keeping the bacterial pellet away from the heat. [7] |
| Contamination occurs during handling. | Maintain strict aseptic technique throughout the procedure. [12] |
| Insufficient broth is added, leading to incomplete resuspension. | Ensure at least 0.3 mL sterile broth is used for proper resuspension. [5] |
| Viability is lost during shipping or storage. | Repeat the procedure with a fresh lyophilized sample if necessary. [3] |

### GLYCEROL STOCK

Objective - To prepare for long-term storage of E. coli at -80 °C using glycerol as a cryoprotectant. Storing bacteria in glycerol stock preserves it for potentially years, preventing cell death and denaturing.

**Materials & Equipment**

- 1.5 mL microcentrifuge tube or screwcap tube
- 100% glycerol (to be diluted with ddH2O to 50%)
- E. coli S17-1 culture grown in broth
- Micropipettes and sterile tips
- Dry ice (for snap freezing, optional)
- -80 °C freezer

**Protocol**

1. Prepare 50% glycerol by diluting 100% glycerol with ddH2O.
2. In a sterile microcentrifuge tube/screwcap tube, add 500 µL of 50% glycerol and 500 µL of bacterial culture.
3. Mix gently by pipetting up and down (avoid bubbles).
4. (Optional) Snap-freeze the vial using dry ice.
5. Store the glycerol stock at -80 °C. [1]

**Troubleshooting & Failures**

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| Cells do not survive freezing | Incorrect glycerol concentration (too low → insufficient protection, too high → toxic). | Ensure final glycerol concentration is ~25% (mix 500 µL of 50% glycerol with 500 µL of culture). [1] |
| Cells are in the stationary phase. | Use a fresh, healthy culture in the mid-logarithmic phase for freezing. [10] |
| Glycerol stock is contaminated | Improper aseptic technique. | Maintain strict aseptic technique when mixing and aliquoting. [12] |
| Stock loses viability over time | Repeated freeze-thaw cycles. | Aliquot stocks into smaller volumes to avoid repeated thawing. [13] |

### REVIVING GLYCEROL STOCK

Objective -To recover viable bacterial cells from -80 °C glycerol stocks and establish active cultures for experimental use. This step provides a consistent starting material and ensures strain integrity for downstream applications.

**Materials & Equipment**

- Glycerol stock (from -80 °C freezer)
- LB agar plates
- LB broth
- Sterile culture tubes
- Pipettes and sterile tips
- Sterile toothpicks or inoculating loops
- Shaking incubator (37 °C, 250 rpm)
- Standard incubator (37 °C)

**Protocol**

1. Retrieve glycerol stock from -80 °C and keep it on ice.
2. Use a sterile loop, pipette tip, or toothpick to take a small sample from the stock.
3. Perform quadrant streaking on an LB agar plate.
4. Incubate the plate overnight at 37 °C.
5. The next day, check for isolated single colonies.
6. Fill a sterile culture tube with LB broth.
7. Use a sterile pipette tip, toothpick, or loop to pick the chosen colony.
8. Transfer the colony into the LB broth.
9. Incubate the culture overnight at 37 °C with shaking at 250 rpm. [28]

**Troubleshooting & Failures**

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| No growth on LB agar plate | Sample is taken improperly from glycerol stock (too little or not sterile). | Use a sterile loop, tip, or toothpick and take a small, sufficient sample from the glycerol stock. [1] |
| No isolated colonies | Overcrowding or streaking is done poorly. | Use a sterile loop, tip, or toothpick and take a small, sufficient sample from the glycerol stock. [11] |
| Contamination during revival | Non-sterile technique. | Maintain aseptic technique throughout the revival process. [12] |
| Broth culture fails to grow | The colony picked is non-viable. | Pick a well-isolated, healthy colony from the agar plate. [13] |
| Uneven growth in broth | Insufficient mixing of inoculum. | Mix gently after transferring the colony into broth. |
| Culture grows slowly | Incubation temperature or shaking is inadequate. | Incubate at 37 °C with proper shaking (250 rpm) for optimal growth. |

### PREPARATION OF CHEMICALLY COMPETENT CELLS

Objective - To prepare E. coli S17-1 cells for transformation by making them chemically competent with CaCl2 treatment. This step increases cell membrane permeability and facilitates efficient uptake of plasmid DNA in subsequent experiments.

**Materials & Equipment**

- Overnight culture of E. coli S17-1
- LB broth
- Pre-chilled 1.5 mL microcentrifuge tubes
- 10% CaCl2 solution (ice cold)
- 15% glycerol + 1.11 g CaCl2 solution (for glycerol stock)
- Spectrophotometer (OD600 measurement)
- Shaking incubator (37 °C, 200 rpm)
- Refrigerated centrifuge
- Ice bucket

**Protocol**

1. Inoculate 0.6 mL of the overnight culture into 12 mL LB broth.
2. Incubate at 37 °C with shaking (200 rpm) for 4 hours.
3. Measure OD600 and harvest when the culture reaches ~0.4-0.6.
4. Place the culture on ice for 10 minutes.
5. Transfer 1 mL aliquots into pre-chilled 1.5 mL microcentrifuge tubes.
6. Centrifuge at 5000 x g for 10 minutes at 4 °C. Discard the supernatant.
7. Resuspend the pellet in 1 mL ice-cold 10% CaCl2. Incubate on ice for 1 hour.
8. Centrifuge at 4000 x g for 15 minutes at 4 °C. Discard the supernatant.
9. Resuspend the pellet in 100 µL ice-cold 10% CaCl2 for immediate heat-shock transformation.
10. Alternatively, resuspend in 100 µL of 15% glycerol + CaCl2 solution for long-term storage at -80 °C. [29][30]

### HEAT SHOCK TRANSFORMATION

Objective -To introduce foreign DNA (plasmid) into competent E. coli cells by temporarily making their cell membranes permeable, allowing the DNA to enter and produce transformed bacteria.

**Materials & Equipment**

- Competent E. coli cells (pre-chilled)
- Resuspended DNA (plasmid or insert)
- 1.5 mL microcentrifuge tubes (pre-chilled)
- Ice and ice bucket
- Pipettes and sterile tips
- Water bath or heat block (42 °C)
- SOC or LB media (pre-warmed to room temperature)
- Sterile Petri plates with appropriate agar and antibiotic
- Sterile glass spreaders or glass beads
- Shaking incubator (37 °C, 200-300 rpm)
- Centrifuge capable of 6800 x g
- Sterile loop or toothpick for picking colonies
- Colony PCR reagents and equipment (if verifying transformants)
- Glycerol for making stocks (optional)

**Protocol**

1. Thaw competent cells on ice.
2. Pipette 50 µL of competent cells into a 1.5 mL tube (pre-chilled).
3. Pipette 1 µL of resuspended DNA into a 1.5 mL tube: pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
4. Close 1.5 mL tubes, incubate on ice for 20-30 minutes: tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
5. Heat-shock tubes at 42 °C for 30-45 seconds.
6. Incubate on ice for 5 minutes.
7. Pipette 950 µL LB media into each transformation: LB media is stored at 4 °C, but can be warmed to room temperature before use. Check for contamination.
8. Incubate at 37 °C for 1 hour, shaking at 200-300 rpm.
9. Take the plate out of the fridge to warm to room temperature.
10. Pipette 100 µL of each transformation onto LB plates. Spread with a sterilized spreader or glass beads immediately.
11. Spin down cells at 6800 x g for 3 minutes.
12. Discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto Petri plates. Spread with a sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower-concentration DNA samples.
13. Incubate transformations overnight (14-18 hr) at 37 °C: incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow, and the antibiotics may start to break down: untransformed cells begin to grow.
14. Pick single colonies: pick single colonies from the transformation. Perform colony PCR to verify part size, make a glycerol stock, grow up cell cultures, and miniprep. [16]

**Troubleshooting & Failures**

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| No colonies on antibiotic plates, but growth in antibiotic broth | Agar plates contain the wrong antibiotic or the wrong concentration. | Verify antibiotic identity and concentration used for plates. [18] |
| Plates have lost potency (the antibiotic has degraded or expired). | Check plate expiry and storage conditions; make fresh plates if unsure (LB agar plates typically remain usable for about 3 weeks at 4 °C; LB broth about 1-1.5 months at 4 °C). [19] |
| Plates have lost potency (the antibiotic has degraded or expired). | Include at least a 1 hr recovery at 37 °C before plating. [20] |
| Plates have lost potency (the antibiotic has degraded or expired). | Let plates warm to room temperature and dry slightly before plating; use sterile spreaders. [21] |
| Plasmid's resistance marker has not been expressed quickly; cells survive in liquid but cannot form colonies on agar immediately. | Plate after recovery, and also plate a portion after a short regrowth (e.g., grow 30-60 minutes in SOC, then plate). [22][20][23] |
| Streak bacteria from the antibiotic broth onto a fresh antibiotic plate to confirm true resistance. [20] |
| No colonies on any plate | Competent cells lose competency (thawed/handled incorrectly; multiple freeze-thaw cycles). | Use freshly prepared or commercially competent cells; avoid freeze-thaw cycles; keep cells on ice. [24] |
| Incorrect heat-shock conditions. | Heat-shock cells at 42 °C for 40 seconds, then immediately place on ice; ensure equipment is calibrated. [20] |
| Incorrect heat-shock conditions. | Check DNA on a gel or Nanodrop; use moderate amounts (1-5 µL ≤ ~50 ng). [20] |
| Very few colonies | Competent cells have low efficiency. | Run a positive control transformation using another known plasmid (e.g., previously confirmed to transform successfully in your lab) to calculate efficiency; if <106 cfu/µg, prepare new competent cells. [25] |
| DNA is too dilute or impure. | Purify DNA before use; avoid salts or ligase buffer carry-over; use ≤5 µL of ligation mix per 50 µL competent cells; check DNA quality on gel/Nanodrop. [26] |
| Recovery time is too short or no shaking during recovery. | After heat shock, add 450-500 µL SOC and recover cells at 37 °C with shaking (200-250 rpm) for 45-60 minutes before plating. [22] |

### EXTRACTION OF PLASMID DNA (MINIPREP)

Objective -To isolate and purify plasmid DNA from bacterial cells using alkaline lysis followed by silica resin column purification. The resulting pure, high-quality plasmid DNA can then be used for a variety of critical downstream applications.

**Materials & Equipment**

- Cultured bacterial solution
- Genedirex Plasmid miniPREP Kit (Cat No. NA005-0100):
→ Buffer S1 with RNase (final conc. 0.1 mg/mL)
→ Buffer S2
→ Buffer S3
→ Spin column
→ Buffer W1
→ Buffer W2 (with ethanol)
→ Sterile ddH2O (pH 7.0-8.5) or Buffer BE (Tris-based elution buffer)

**Protocol**

**1 Bacterial Cells Harvesting**
1. Transfer the bacterial culture (up to 1.5 mL) to a microcentrifuge tube.
2. Centrifuge at 14,000 x g for 1 minute and discard the supernatant.
**Step 2 Resuspension**
1. Resuspend the pellet of bacterial cells in 200 μL of Buffer S1 (RNase A added).
**Step 3 Lysis**
1. Add 200 μL of Buffer S2 and mix thoroughly by inverting the tube 10 times (do not vortex). Stand at room temperature for 2 minutes or until the lysate is homogeneous.
**Step 4 Neutralization**
1. Add 300 μL of Buffer S3 and mix immediately and thoroughly by inverting the tube 10 times (do not vortex).
2. Centrifuge at 14,000 x g for 3 minutes.
**Step 5 Binding**
1. Place a PM column in a Collection Tube. Apply the supernatant (from step 4) to the PM column by decanting or pipetting.
2. Centrifuge at 14,000 x g for 30 seconds, discard the flow-through, and place the PM column back into the same Collection Tube.
**Step 6 Wash**
1. Add 400 μL of Buffer W1 into the PM column.
2. Centrifuge at 14,000 x g for 30 seconds.
3. Discard the flow-through and place the PM column back into the same Collection Tube.
4. Add 600 μL of Buffer W2 (ethanol added) into the PM column.
5. Centrifuge at 14,000 x g for 30 seconds.
6. Discard the flow-through and place the PM column back into the same Collection Tube.
7. Centrifuge at 14,000 x g again for 2 minutes to remove the residual Buffer W2.
**Step 7 Elution**
1. Place the PM column in a clean 1.5 mL microcentrifuge tube. Add 50-200 μL of Buffer BE or H2O (pH 7.0-8.5) to the center of the PM column. Let it stand for 2 minutes and centrifuge at 14,000 x g for 2 minutes.
2. Take the flow-through, add it again to the center of the PM column, and centrifuge at 14,000 x g for 2 minutes. [4]

### RESUSPENSION OF THE SAMPLE FROM THE DISTRIBUTION KIT

Objective - To resuspend dried DNA samples from the distribution kit into solutions for subsequent transformation into competent cells. By properly rehydrating the DNA (containing cresol red dye for visual confirmation), we ensure accurate handling and avoid cross-contamination. The resuspended DNA is then used for bacterial transformation, colony growth, minipreparation, and glycerol stock creation, enabling reliable downstream applications such as restriction digest and sequencing.

**Materials & Equipment**

- Distribution kit DNA plate (dried DNA samples with cresol red dye)
- ddH2O (pH 7.0)
- Pipettes and sterile tips
- Sterile 1.5 mL microcentrifuge tubes

**Protocol**

Note: There is an estimated 2-3 ng of DNA in each well. When following this protocol, assume that you are transforming with 200-300 pg/µL.

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that is needed. Ensure the plate is properly oriented. Do not remove the foil cover, as doing so can lead to cross-contamination between the wells.
2. Pipette 10 µL of dH2O (distilled water) into the well. Pipette mix a few times and let sit for at least 5 minutes to ensure the dried DNA is fully resuspended. The resuspension appears red, as the dried DNA contains cresol red dye. [14]

### PREPARATION OF X-GAL LB AGAR PLATES FOR BLUE/WHITE COLONY SCREENING.

Objective - To prepare LB agar plates with X-Gal for blue/white colony screening of recombinant bacterial colonies.

**Materials & Equipment**

- LB agar
- Petri dish
- Bunsen Burner
- Thermo Scientific X-Gal
- Hockey Stick

**Protocol**

1. Pour sterile warm LB agar (about 25 mL) into a Petri dish.
2. Dry opened LB plates at room temperature around bunsen burner for about 30 minutes.
3. Add 40 μL of the Thermo Scientific X-Gal Solution (20 mg/mL), ready-to-use (Cat #R0941).
4. Spread evenly on the plate with a Hockey stick.
5. Dry open LB plates at room temperature around the Bunsen burner for about 30 minutes. [35]

### CONJUGATION(SOLID CULTURE)

Objective - To perform solid surface conjugation between E. coli S17-1 (carrying BLIP-I or BLIP-II) and E. coli DH5α (Ampicillin-resistant), allowing horizontal gene transfer through cell-to-cell contact on LB agar. This experiment evaluates the effectiveness of BLIP-I and BLIP-II in inhibiting β-lactamase activity after conjugation.

**Materials & Equipment**

Bacterial Strains

- Donor: E. coli S17-1 carrying BLIP-I or BLIP-II (Chloramphenicol-resistant)
- Recipient: E. coli DH5α (Ampicillin-resistant)

Selective agar plates:

- LB + Ampicillin+X-gal (50 µg/mL)
- LB + Chloramphenicol+X-gal (25 µg/mL)
- LB + Ampicillin + Chloramphenicol+X-gal
- PBS buffer (pH 7.4) – for washing and resuspension steps

DAP (optional) – added only if donor requires diaminopimelic acid for growth
Equipment and Consumables

- Microcentrifuge tubes (1.5 mL)
- Micropipettes and sterile tips
- Centrifuge (capable of ~6800 x g)
- Spectrophotometer (for OD600 measurement)
- Sterile inoculating loops or pipette tips (for mixing donor and recipient on plate)
- Vortex mixer
- 37 °C incubator

**Protocol**

**Step 1. Prepare overnight cultures:**
1. Grow E. coli S17-1 (BLIP-I / BLIP-II) and E. coli DH5α (Ampicillin-resistant) separately in LB broth at 37 °C, shaking at 250 rpm overnight until visibly turbid.
**Step 2. Remove residual antibiotics:**
1. Centrifuge 1 mL of each overnight culture at ~6800 x g for 1 minute, then discard the supernatant.
**Step 3. Wash cells with PBS:**
1. Resuspend each pellet in 1 mL PBS, pipette up and down to mix, centrifuge again, and discard the supernatant. Repeat once to ensure all antibiotics are removed.
**Step 4. Normalize cell densities:**
1. Resuspend each pellet in 500 µL PBS and measure OD600. Adjust cell densities to approximately equal levels for a balanced donor-recipient ratio.
**Step 5. Mix donor and recipient:**
1. In a sterile microcentrifuge tube, mix donor and recipient cells in a 1:1 ratio (total volume ≈ 100 µL). Mix gently by pipetting.
**Step 6. Spot mixture onto the plate:**
1. Spot 50–100 µL of the mixture onto an LB agar plate without antibiotics (with DAP if required). Do not spread; allow the spot to air-dry under sterile conditions.
**Step 7. Incubate for conjugation:**
1. Incubate the plate at 37 °C for 4–6 hours to allow cell-to-cell contact and plasmid transfer.
**Step 8. Recover conjugation mixture:**
1. After incubation, scrape the bacterial spot into 1 mL PBS and vortex briefly to resuspend.
**Step 9. Plate on selective media:**
1. Dilute the suspension as needed and plate onto LB + Ampicillin + Chloramphenicol selective agar to isolate transconjugants.
**Step 10. Incubate and observe:**
1. Incubate selective plates overnight at 37 °C. Colonies confirming successful conjugation can be observed the next day. [32][33][34]

**Troubleshooting & Failures**

| Problem | Possible Reason | Solution |
| --- | --- | --- |
| No colonies observed after conjugation | Low donor or recipient activity during mixing (using old or stationary-phase cultures reduces conjugation efficiency). | Use fresh overnight cultures (OD600 ≈ 0.4-0.6) for both donor and recipient. [32] |
| Weak or no growth on selective (Chl) plates | Incorrect antibiotic concentration or incomplete mixing of donor and recipient cells. | Confirm chloramphenicol working concentration (25 µg/mL) and ensure firm contact between the two strains when mixing on the LB plate. |
| Overgrowth of donor strain on selection plate | S17-1 has intrinsic resistance to chloramphenicol, which can mask DH5α transconjugants. | Use a differential screening method (e.g., fluorescence marker or colony PCR) to verify true DH5α transconjugants. |
| Contamination or uneven growth on conjugation plate | Insufficient sterilization of loops or incubator humidity. | Use sterile tips for mixing and ensure all plates are parafilm-sealed to prevent airborne contamination. |

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    - *How to Make Chemically Competent E. coli.* Youtu.be. <https://youtu.be/H4X-5Bg0bE8?si=hEPtQC9LKfNHRjSu> (accessed 2025-09-13)
    - *How to Make Competent Cells: Protocols and Tips.* Zymo Research International. <https://zymoresearch.eu/blogs/blog/how-to-make-competent-cells-protocols-and-tips?srsltid=AfmBOorWrTUbyt60IlevDWfroiJWyt5QnEfbkKV-RtjWFN9gNSiDxc4Z> (accessed 2025-09-13)
    - *No Bands - Genotyping.* The Jackson Laboratory. <https://www.jax.org/jax-mice-and-services/customer-support/technical-support/genotyping/troubleshooting/no-bands>
    - *Barrick Lab: Protocols Conjugation.* barricklab.org. <https://barricklab.org/twiki/bin/view/Lab/ProtocolsConjugation>
    - *Introductory Bacterial Conjugation TEACHER'S MANUAL and STUDENT GUIDE Strain I Streptomycin-Resistance Gene on Chromosome Strain II Ampicillin-Resistance Gene on Plasmid.* [PDF Link](https://www.pltw.org/hubfs/operations/equipment_supplies/bms_Bill_of_materials/211125_Introductory%20Bacterial%20Conjugation_Manual.pdf)
    - Elston, K. M.; Phillips, L. E.; Leonard, S. P.; Young, E.; Holley, J. C.; *Tasneem Ahsanullah; McReynolds, B.; Moran, N. A.; Barrick, J. E. The Pathfinder Plasmid Toolkit for Genetically Engineering Newly Isolated Bacteria Enables the Study of Drosophila-Colonizing Orbaceae.* ISME Communications 2023, 3 (1). <https://doi.org/10.1038/s43705-023-00255-3>
    - *Preparation of X-Gal/IPTG LB Agar Plates for Blue/White Colony Screening.* [https://documents.thermofisher.com/TFS-Assets/BID/Methods-&-Protocols/75-preparation-of-x-gal-iptg-lb-agar-plates-for-blue-white-colony-screening.pdf](https://documents.thermofisher.com/TFS-Assets/BID/Methods-%26-Protocols/75-preparation-of-x-gal-iptg-lb-agar-plates-for-blue-white-colony-screening.pdf)(accessed 2025-10-08).](https://www.edvotek.com/transformation-troubleshooting-guide?srsltid=AfmBOopbC9MGzTWFGoqM00zpx427wpjxpz9H5_qTv4Dv1hkbK2PNQr74&)

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Defy
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{% block title %}Wet Lab - Results{% endblock %}
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[A NOTE ON OUR JOURNEY](#overview)
[CONSTRUCTION OF PLASMID 1](#construction_of_plasmid_1)
[CONSTRUCTION OF PLASMID 2](#construction_of_plasmid_2)
[MINIPREP OF BBA\_J435320](#miniprep_of_bba)
[DONOR BACTERIA](#donor_bacteria)
[RECIPIENT BACTERIA](#recipient_bacteria)
[CONJUGATION](#conjugation)
[EXPECTED OUTCOME PATTERNS](#expected_outcome_patterns)
[FINAL RESULTS](#final_results)
[TIME CONSTRAINTS AND LIMITATIONS](#time_constraints_and_limitations)
[ADDITIONAL PLAN](#additional_plan)
[FINAL WORDS](#final_words)

![Top Image](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/lightbluetitlebg.png)

# Results

## A NOTE ON OUR JOURNEY

As a first-year iGEM team, all of our members dedicated themselves to tackling the urgent challenge of antimicrobial resistance (AMR). However, our journey was full of ups and downs. With limited experience in wet lab work, restricted access to lab equipment, and a school schedule that often left us with little lab time, we faced many obstacles. On top of that, our lab was not always available, and as a brand-new team, we were constantly encountering experimental processes and unexpected outcomes that were entirely new to all of us. Despite all that, we still gave our very best, pouring in our time and effort just to bring our ideas to life in the lab. Even though we did not manage to finish every experiment, what we gained was just as valuable. The experience of trying, learning, and growing together as a team. The setbacks did not stop us, they reminded us why we started, and gave us the determination to keep moving forward.

## CONSTRUCTION OF PLASMID 1 (DONOR PLASMID)

To construct our donor plasmid, we used the pSB1C3 backbone with mCherry and Chloramphenicol resistance as the base. Into this backbone, we aimed to insert the BLIP-I and BLIP-II sequences, allowing the plasmid to serve as a carrier of BLIP in our donor bacterium. This construct would later enable us to test conjugation and observe whether BLIP can inhibit β-lactamase activity in recipient strains.

Step 1. Sequence optimization

To ensure reliable expression of our BLIP constructs in E. coli, we reverse-translated the protein sequences of BLIP-I and BLIP-II obtained from the NIH database and performed codon optimization specifically for E. coli expression. The resulting gene sequences were adjusted to satisfy the codon usage and sequence complexity requirements of GenScript and Twist Bioscience, while preserving the original amino acid sequences. **Figure 1** presents the finalized nucleotide sequences submitted for gene synthesis following optimization.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f1.png)

**Figure 1.** Sequence optimization for gene synthesis

Step 2. PCR amplification + Gel purification

The BLIP gene fragments were successfully amplified by PCR, producing clear bands of the expected size on agarose gel electrophoresis (**Figure 2**). The amplified products were then excised and purified using the GeneDireX PCR Clean-Up and Gel Extraction Kit to remove residual primers, nucleotides, and enzymes that could interfere with downstream reactions. (**Figure 3**)

After purification, the resulting high-purity DNA was directly used for restriction digestion with EcoRI and PstI to prepare the inserts for ligation into the pSB1C3 backbone. This purification step ensured clean, intact DNA suitable for cloning and efficient enzymatic digestion.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f2.png)

**Figure 2.** Gel electrophoresis results of the PCR amplifications of BLIP-I and II

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f3.png)

**Figure 3.** Purification of PCR products by excision and gel purification kit

Step 3. Transformation of DH5ɑ with PSB1C3-mCherry (distribution kit #O11)

To begin constructing our donor plasmid, we first transformed E. coli DH5α with the pSB1C3-mCherry backbone (distribution kit #O11). This backbone carried Chloramphenicol resistance and the mCherry reporter, which would later allow us to track and confirm our constructs visually. When we checked the transformation plates, only a single colony appeared.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f4.png)

**Figure 4.** Resuspended pSB1C3-mCherry backbone (distribution kit #O11)

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f5.png)

**Figure 5.** Transformation result of DH5ɑ\_PSB1C3\_mCherry. The outgrowth culture was plated on a Ch-LB plate and cultured at 37 ℃ overnight.

Step 4. Subculturing the DH5ɑ\_PSB1C3\_mCherry

After we transformed DH5α with pSB1C3-mCherry, we were excited to see a colony appear, but also nervous, because it was the only colony on the plate. Losing it would mean starting over from scratch. To avoid this risk, we first streaked the colony onto a Ch-LB plate to expand our supply. We also inoculate it to a LB broth to allow it to grow without antibiotic pressure. Once it produced more colonies, we subcultured it onto both a Ch-LB plate and a Ch-LB plate . This way, we could maintain selective pressure while also ensuring we didn’t lose our only positive clone.

This careful step gave us enough material to continue with plasmid preparation and preserved our successful transformation for the next stages of plasmid construction.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f6.png)

**Figure 6.** Subculture result of DH5ɑ\_PSB1C3\_mCherry in Ch-LB plate

Step 5. Making glycerol stocks of DH5ɑ\_PSB1C3\_mCherry

Once we confirmed that our DH5α\_pSB1C3\_mCherry clone was stable, we prepared glycerol stocks to preserve it. Since this plasmid was the backbone for our donor construction, we wanted to make sure we would not lose the strain after future subculturing or experiments. By freezing glycerol stocks at –80 °C, we created a long-term, reliable source of our construct that we could return to at any time. This step gave us security and ensured that even if future transformations failed, we could always recover our starting material.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f7.png)

**Figure 7.** Glycerol stocks of DH5ɑ\_PSB1C3\_mCherry

Step 6. Miniprep of PSB1C3\_mCherry

After successfully growing DH5α carrying the pSB1C3-mCherry backbone, we performed a plasmid miniprep to extract and purify the plasmid DNA. This step was essential because it provided us with a clean supply of the backbone needed for our ligation with BLIP-I and BLIP-II. Having purified DNA not only allowed us to move forward with restriction digestion but also gave us a reliable stock that could be used repeatedly without depending on fresh bacterial cultures each time.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f8.png)

**Figure 8.** Pellet of bacteria collected during the centrifugation step in the miniprep protocol

Step 7. Restriction digestion

With purified pSB1C3-mCherry plasmid DNA (from step 6) and BLIP genes (step 2) in hand, we carried out restriction digestion with EcoRI and PstI to prepare the backbone for ligation with BLIP-I and BLIP-II inserts. Our initial digests produced very faint or sometimes missing bands on the gel, and the DNA concentrations after purification were often too low for efficient cloning. (**Figure 9**)

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f9.png)

**Figure 9.** Gel electrophoresis of restriction digestion products of purified DNA samples using the initial purification protocol. Weak DNA band intensity indicates low recovery efficiency when ddH2O was used for elution from the silica column.

Before identifying the main cause of our low DNA recovery, we conducted several troubleshooting steps to improve yield during gel extraction and purification.

First, we verified the preparation of all reagents and discovered that ethanol had not been added to Buffer W2, a crucial component for DNA precipitation and binding to the silica column. After correcting this oversight, we observed only slight improvement in DNA retention. Second, we increased the DNA loading concentration for gel electrophoresis by combining multiple samples into a single wide well to visualize faint bands more clearly. Third, we attempted back-elution of the silica column to recover additional DNA that might have remained bound after the initial elution.

However, none of these adjustments significantly improved recovery. The DNA yield remained low, and gel bands were still faint. It was only then that we considered the potential effect of pH on DNA elution. We realized that ddH₂O gradually becomes slightly acidic over time (**Figure 10**), which compromises DNA stability and recovery efficiency.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f10.png)

**Figure 10.** The ddH2O used for the elution was found to be too acidic. According to the manual, the optimal pH for the eluent should be around 7.0~8.5

By switching to Tris buffer for elution, we achieved clearer digested bands and substantially higher DNA yield. (**Figure 11**) This inspiring result marked an important **engineering success** in our workflow. The adjustment provided sufficient high-quality backbone DNA for the next steps of our cloning process. The gels containing the desired DNA fragments were carefully excised, extracted, and purified using the GeneDireX Gel Extraction Kit. The resulting purified DNA samples were then used directly in the subsequent ligation and transformation steps.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f11.png)

**Figure 11.** Gel electrophoresis of the restriction digestion results using purified BLIP-I & II sequences and pSB1C3 backbone (labelled as C) using modified protocol (Tris buffer elution).

Step 8. Ligation and transformation of DH5ɑ with PSB1C3\_BLIP-I and PSB1C3\_BLIP-II

After preparing both our BLIP inserts and the pSB1C3-mCherry backbone, we performed ligation to assemble the donor plasmids and transformed them into E. coli DH5α.

At first, the transformation did not yield colonies after standard overnight incubation. (**Figure 12**) When reviewing our reference protocol provided by NYCU Formosa, we realized that the amount of plasmid DNA we used during transformation was too low, 3 µL instead of the intended 10 µL, which likely reduced the efficiency of the transformation. Instead of discarding the attempt, we decided to extend incubation to 36 hours in hopes of recovering transformants.

This patience paid off. After the extended incubation, one colony began to appear on the Ch-LB plate spread with DH5α\_pSB1C3\_BLIP-I-mCherry. (**Figure 13**) The colony showed the distinct pink coloration from the mCherry reporter, which we affectionately referred to as our “Pinky Juniors.”

For DH5α\_pSB1C3\_BLIP-I-mCherry, no colony showed up on the Ch-LB plates even after prolonged incubation; however, the remaining outgrowth culture we added to the Ch-LB broth showed bacterial growth. (**Figure 14**)

The fluorescence confirmed that our ligations had succeeded and that DH5α was now carrying the BLIP constructs. This was a critical milestone: despite setbacks, we demonstrated that both BLIP-I and BLIP-II could be successfully cloned into our donor backbone and maintained in bacteria.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f12.png)

**Figure 12.** Transformation result of DH5α\_pSB1C3\_BLIP-I-mCherry (Left) and DH5α\_pSB1C3\_BLIP-II-mCherry (Right). No bacterial growth after spreading the outgrowth culture on Ch-LB plates.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f13.png)

**Figure 13.** Transformation result of DH5α\_pSB1C3\_BLIP-I-mCherry

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f14.png)

**Figure 14.** Transformation result of DH5α\_pSB1C3\_BLIP-II-mCherry. Bacterial growth in Ch-supplemented LB broth.

Step 9. Subculturing of DH5ɑ\_PSB1C3\_BLIP-I and DH5ɑ\_PSB1C3\_BLIP-II

After the breakthrough of obtaining colonies carrying our BLIP constructs, our next priority was to secure and expand them. We carefully picked colonies of DH5α\_pSB1C3\_BLIP-I and DH5α\_pSB1C3\_BLIP-II and subcultured them into fresh Ch-LB broth and onto selective plates.

This step ensured that our precious transformants would not be lost and gave us enough material for downstream experiments. Watching the pink-fluorescent cultures grow stronger was a reassuring sign that our plasmids were stable and ready for further testing. By establishing these subcultures, we created reliable working stocks of both BLIP-I and BLIP-II donor plasmids, marking an important checkpoint in our construction process.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f15.png)

**Figure 15.** Subculture of DH5α\_pSB1C3\_BLIP-I-mCherry

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f16.png)

**Figure 16.** Subculture of DH5α\_pSB1C3\_BLIP-II-mCherry

Step 10. Miniprep of pSB1C3\_BLIP-I and pSB1C3\_BLIP-II

With stable subcultures of DH5α carrying our BLIP constructs, we performed a plasmid miniprep to extract and purify the DNA. This step was crucial, as it provided us with a clean and concentrated supply of the pSB1C3\_BLIP-I and pSB1C3\_BLIP-II plasmids for further use.

The purified plasmids were intended for transformation into our donor strain (E. coli S17-1), where they would act as the vehicles for conjugation experiments. By completing this step, we successfully created ready-to-use BLIP plasmids that could be stored, shared, and tested in our conjugation system.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f17.png)

**Figure 17** Miniprepped pSB1C3\_BLIP-I and pSB1C3\_BLIP-II plasmids

## CONSTRUCTION OF PLASMID 2 (RECIPIENT PLASMID)

To test the effect of our BLIP donor plasmids, we needed a recipient plasmid carrying ampicillin resistance. This plasmid would serve as the counterpart in our conjugation system, allowing us to observe whether BLIP could inhibit β-lactamase and resensitize resistant bacteria.

Step 1. Resuspension of samples from the distribution kit.

To begin constructing our recipient plasmid, we first resuspended several samples from the iGEM distribution kit, including BBa\_J435300, BBa\_J43550, and BBa\_J435320. These plasmids carried different versions of ampicillin resistance, and we prepared them so they could be used directly in our transformation experiments. By having these plasmids ready, we ensured that the next steps, transforming E. coli DH5α, would proceed without delays.

Interestingly, while the iGEM distribution kit resuspension is normally expected to turn red due to the cresol red dye, our samples appeared brownish immediately after resuspension. This unexpected color shift was unusual and suggested that the dye or plasmid preparation may have behaved differently from what we anticipated.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f18.png)

**Figure 18.** Resuspended Product of BBa\_J435300 from Distribution Kit #G7, Plate #2

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f19.png)

**Figure 19.** Resuspended Product of BBa\_J43550 from Distribution Kit #O6, Plate #2

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f20.png)

**Figure 20.** Resuspended Product of BBa\_J435320 from Distribution Kit #O9, Plate #2

Step 2. Transformation of DH5ɑ with BBa\_J435300 (Distribution Kit #G7, Plate #2)

We attempted to transform E. coli DH5α with BBa\_J435300, the plasmid carrying ampicillin resistance, to create our recipient bacteria. This step was essential for building the counterpart in our conjugation system.

However, the transformation did not succeed. After reviewing the process, we suspected that the issue may have come from the resuspended distribution kit sample, which had been stored in the freezer for too long. Despite this setback, the attempt helped us identify potential sources of error and prepared us for our subsequent transformations.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f21.png)

**Figure 21.** Transformation of DH5ɑ with BBa\_J435300

Step 3. Mistaken transformation of DH5ɑ with BBa\_J43550 (Distribution Kit #O6, Plate #2)

The plasmid from O6 instead of O9. This plasmid, BBa\_J43550, carried an ampicillin resistance backbone but was originally designed for use in yeast. Surprisingly, after transformation into E. coli DH5α, we observed growth on our plates. At first, this result left us puzzled, since we had not expected a yeast-related plasmid to support growth in a bacterial system.

Curious about this unexpected outcome, we looked up the plasmid's details online and discovered that while it indeed contained an ampicillin resistance gene, this alone could not explain why it persisted in E. coli. The most likely explanation is that BBa\_J43550 also carries an E. coli-compatible origin of replication, which allowed it to replicate in DH5α. Combined with the AmpR gene, this enabled colonies to survive and expand.

This accidental transformation not only surprised us but also taught us the importance of focus and accuracy in every step of the lab process. What began as a small oversight became an opportunity to understand plasmid compatibility more deeply and reminded us that careful attention to detail is essential in synthetic biology.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f22.png)

Figure 22. Transformation of DH5ɑ with BBa\_J43550

Step 4. Transformation of DH5ɑ with BBa\_J435320 (The Correct Distribution Kit #O9, Plate #2)

We then performed the transformation using the correct recipient plasmid, BBa\_J435320 (O9). Initially, we saw no colonies on the LB agar plate with ampicillin, which suggested that the transformation had failed. (**Figure 23**) However, when we checked the LB broth with ampicillin where the transformants had also been cultured, we observed visible growth. (**Figure 24**) This result indicated that the transformation had, in fact, worked, just at a very low efficiency that did not immediately appear on plates.

To further confirm the transformation, we plated the culture again onto fresh ampicillin plates, and this time, colonies successfully appeared. We then streaked these colonies for single isolates to prepare them for subculturing.

We also noted that the overall growth rate of our DH5α transformants was slower than expected. Based on repeated observations across different transformations, we suspected that this was due to the low quality of our DH5α competent cells, which had gone through stressful shipping and handling. This may have weakened the cells and reduced their transformation efficiency, leading to delayed or reduced colony formation.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f23.png)

**Figure 23.** Transformation of DH5ɑ with BBa\_J435320 on Amp-LB plate

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f24.png)

**Figure 24.** DH5α \_BBa\_J435320 grown in Amp-LB broth. The third and fourth tubes, inoculated with concentrated cells following heat-shock transformation, exhibit visible turbidity, indicating successful bacterial growth.

MINIPREP OF BBA\_J435320 (DISTRIBUTION KIT #O9, PLATE #2)

Miniprep of BBa\_J435320 (Distribution Kit #O9, Plate #2)

After confirming growth of the O9 transformants, we performed a miniprep to extract plasmid DNA. This step was essential to obtain a clean and concentrated supply of the recipient plasmid, which would later be used in our conjugation experiments. The purified plasmid ensured that we could move forward without depending solely on live cultures.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f25.png)

**Figure 25** Miniprepped BBa\_J435320 plasmids

## DONOR BACTERIA: S17-1\_PSB1C3\_BLIP-I AND S17-1\_PSB1C3\_BLIP-II

For the donor part of our conjugation system, we chose E. coli S17-1, a strain well-suited for plasmid transfer because of its built-in conjugation functions. Into this strain, we aimed to introduce our constructed plasmids PSB1C3\_BLIP-I and PSB1C3\_BLIP-II, enabling S17-1 to serve as the vehicle for delivering BLIP into recipient bacteria.

Unlike our familiar DH5α strain, working with S17-1 presented new challenges, especially since we did not have streptomycin available until mid-October and had to maintain the strain without antibiotics under strict aseptic conditions. This marked the beginning of our efforts to establish a functional donor strain for conjugation.

Step 1. Rehydration and initial growth of S17-1

We began by rehydrating the lyophilized E. coli S17-1 strain, carefully reviving it to prepare for downstream use as our donor bacteria.

At this stage, we faced an additional challenge: our lab did not yet have access to streptomycin, which is normally used to maintain S17-1 cultures.

As a result, we had to grow the strain without antibiotics, relying instead on strict aseptic conditions to prevent contamination. This step required extra caution, but it allowed us to successfully establish the initial growth of S17-1 and set the foundation for its role as our donor strain.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f26.png)

**Figure 26.** Initial growth of E. coli S17-1

Step 2. Making glycerol stock

To preserve our revived E. coli S17-1 strain for long-term use, we prepared glycerol stocks and stored them at –80 °C. This ensured that we could always return to a stable, viable culture for future experiments without the need to repeat the rehydration process. By securing these stocks early, we created a reliable backup that safeguarded our progress and gave us confidence to move forward with subsequent transformations.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f27.png)

**Figure 27.** Glycerol stocks of S17-1

Step 3. First attempt of Heat-shock transformation

This was our very first time attempting to transform E. coli S17-1. Without realizing that the cells were not chemically competent, we proceeded with the transformation as if they were ready. Unsurprisingly, the attempt failed, and no colonies were observed on our plates. Although it was disappointing at the moment, this mistake became an important learning experience for us. It taught us the critical role of preparing competent cells before transformation and reminded us that each step in the workflow must be carefully checked before moving forward.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f28.png)

**Figure 28.** Heat-shock transformation of S17-1 with pSB1C3\_BLIP-1\_mCherry (1st attempt)

Step 4. Second attempt of heat-shock transformation

During our first attempt at preparing chemical competent cells from E. coli S17-1, we struggled to obtain a sufficient pellet after centrifugation. This was likely due to too short of an incubation time, which resulted in very low cell density. The pellet we collected was extremely small, and as a result, the cells might not be successfully made competent. In the end, we still decided to store these as glycerol stocks.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f29.png)

**Figure 29.** The extremely small pellet obtained during our first attempt at preparing competent E. coli S17-1 cells

In our second attempt of preparing chemical competent cells, we extended the growth time and managed to obtain a good amount of pellet, indicating healthier biomass. However, instead of using the cells immediately, we stored both attempts as glycerol stocks at –80 °C.

We then used the competent E. coli S17-1 that we had prepared earlier and stored as glycerol stock at –80 °C to perform a heat shock, but it failed to produce any colonies. We suspected that this was due to the S17-1 competent cells losing competency after being frozen for too long. S17-1 appears to be much more sensitive, and using freshly prepared competent cells would likely have increased our success rate.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f30.png)

**Figure 30.** Heat-shock transformation of pSB1C3-BLIP-I into E. coli S17-1

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f31.png)

**Figure 31.** Heat-shock transformation of pSB1C3\_BLIP-II into E. coli S17-1

Step 5. Third attempt of heat-shock transformation

In our third attempt, we prepared E. coli S17-1 competent cells and, instead of storing them, we used them fresh for transformation. This change proved crucial, the freshly prepared competent cells successfully took up the BLIP plasmid, confirming that S17-1 could be transformed once handled under the right conditions. This outcome demonstrated the importance of using fresh competent cells with S17-1, as storage in glycerol stocks appeared to compromise transformation efficiency. By using this approach, we ensured that future heat-shock transformations would not fail due to the use of non-fresh S17-1 cells.

For the plasmid source, we used the plasmids (pSB1C3\_BLIP-I and pSB1C3\_BLIP-II) that had been successfully miniprepped from our DH5α transformants. This DH5α backup was created as mentioned earlier in the “Construction of Plasmid 1” section. By introducing these plasmids into fresh competent S17-1, the transformation was successful, establishing S17-1 as a donor strain for our conjugation experiments.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f32.png)

**Figure 32.** Transformation results of S17-1\_pSB1C3\_BLIP-I-mCherry

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f33.png)

**Figure 33.** Transformation results of S17-1\_pSB1C3\_BLIP-II-mCherry

## RECIPIENT BACTERIA: DH5α\_BBA\_J435320

Step 1. Subculturing of DH5ɑ\_BBa\_J435320 after the transformation

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f34.png)

**Figure 34.** Subculture results of DH5ɑ\_BBa\_J435320

## CONJUGATION

Step 1. Solid Conjugation

We For solid conjugation, we co-cultured the donor strain (E. coli S17-1 carrying pSB1C3-BLIP) and the recipient strain (E. coli DH5α carrying BBa\_J435320) on LB agar plates and LB agar supplemented with X-gal. The plates were only supplemented with X-gal as we could not get IPTG to our lab in time. After incubation at 37 °C for six hours, the plates exhibited visible pink and blue coloration, corresponding to the characteristic pigmentation of the donor (mCherry expression) and the recipient (lacZ expression), respectively. The presence of both colors indicated that both strains were actively growing and in close contact, a necessary condition for conjugation to occur on solid media.began by rehydrating the lyophilized E. coli S17-1 strain, carefully reviving it to prepare for downstream use as our donor bacteria.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f35.png)

**Figure 35.** Solid conjugation result after 6 hours incubation at 37 °C

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f36.png)

**Figure 36.** Solid conjugation result (X-gal added) after 6 hours incubation at 37 °C

Step 2. Validation and Final Results

After several days of preparation, we finally obtained our conjugation results. They worked perfectly and fully supported our hypotheses. Despite our extremely tight schedule (with the wiki freeze just hours away), we were able to capture the key experimental outcomes that confirm our system’s functionality. To provide context for these final results, we first outline the potential outcome patterns we anticipated, followed by our actual observed results.

Following incubation for conjugation, we gently scraped the mixed bacterial growth from the plate surfaces and resuspended the cells in 1 mL of sterile PBS. From this suspension, 100 µL aliquots were plated onto chloramphenicol (Ch), ampicillin (Amp), and dual-antibiotic (Ch + Amp) LB plates, each supplemented with X-gal, and incubated overnight for selection and validation.

To evaluate both conjugation success and BLIP functionality, we combined antibiotic selection with blue-white screening.

EXPECTED OUTCOME PATTERNS

We carefully considered possible plate outcomes to interpret our conjugation data effectively:

- Pink and blue colonies on Ch plates; no growth on Ch + Amp plates — ideal outcome: Complete proof of concept. This pattern would confirm that conjugation successfully transferred the BLIP plasmid from E. coli S17-1 to E. coli DH5α and that BLIP effectively inhibited β-lactamase activity.
- Pink colonies on Ch plates; blue colonies on Amp plates; no growth on Ch + Amp plates — partial or failed conjugation:Indicates no plasmid transfer between donor and recipient. The donor and recipient populations remain separate, and BLIP's inhibitory effect cannot be verified.
- Pink and blue colonies on Ch plates; growth on Ch + Amp plates — successful conjugation, inactive BLIP: Suggests plasmid transfer occurred, but BLIP failed to inhibit β-lactamase activity in the recipient strain.

FINAL RESULTS

We carefully considered possible plate outcomes to interpret our conjugation data effectively:

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f37.png)

**Figure 37.** Conjugation results between E. coli S17-1\_pSB1C3-BLIP-I-mCherry and E. coli DH5α\_BBa\_J435320.
Left: Pink and blue colonies on the Ch-LB plate indicate donor and transconjugant cells.
Middle: Blue colonies on the Amp-LB plate show recipient and transconjugant strains carrying lacZ.
Right: Only one pink colony on the dual Ch-Amp-LB plate demonstrates effective BLIP-mediated inhibition of β-lactamase activity.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f38.png)

**Figure 38.** Conjugation results between E. coli S17-1\_pSB1C3-BLIP-II-mCherry and E. coli DH5α\_BBa\_J435320.
Left: Pink and blue colonies on the Ch-LB plate indicate donor and transconjugant cells.
Middle: Blue colonies on the Amp-LB plate show recipient and transconjugant strains carrying lacZ.
Right: No colonies on the dual Ch-Amp-LB plate demonstrate complete BLIP-mediated inhibition of β-lactamase activity.

TIME CONSTRAINTS AND LIMITATIONS

At the time of the wiki freeze, our final conjugation plates were still incubating overnight, and we had very limited time to conduct additional verification steps. Ideally, we would have miniprepped the transconjugants and performed targeted PCR or restriction digestion to confirm the plasmid sequences. However, due to the rapidly approaching deadline, we were unable to carry out these follow-up validations.

Nevertheless, the observable phenotypes on our selection plates already provide clear functional evidence supporting successful conjugation and BLIP activity, fulfilling our Proof of Concept for the PlasMission Spy system.

## ADDITIONAL PLAN

We began with PCR amplification of BLIP-I and BLIP-II, but the gel electrophoresis showed heavy smearing and low band intensities. (**Figure 39**)The issue was later traced to a primer ordering mistake. As we ran out of VR and VF2 primers, we ordered a new batch; however, instead of the primer sequences (VR and VF2), the primer binding site sequences were mistakenly ordered. In addition, the restriction digestion of BBa\_J435320 produced no visible bands, indicating that the miniprepped plasmid likely contained little or no DNA, possibly due to poor miniprep practice.
By the time these issues were identified, we had successfully constructed the donor strain S17-1\_pSB1C3\_BLIP-1-mCherry for conjugation, making this direct cloning approach unnecessary. The plan was therefore discontinued.

![BLIP-I Structure](https://gitlab.igem.org/2025/hia-taiwan/-/raw/main/wiki/pages/f39.png)

**Figure 39.** Gel electrophoresis result of the PCR products with erroneous VR and VF2 primers

FINAL WORDS

This project has been a truly memorable experience for every single member of our wet lab team. Each of us feels deeply grateful to have had the opportunity to participate in this year’s iGEM competition, to learn, to experiment, and to grow together. Even though our journey was far from perfect—filled with mistakes, unexpected outcomes, and results that did not always align with our expectations, it was these very challenges that made the experience meaningful.

What we gained was more than just data or plasmids, it was the resilience to keep trying, the courage to embrace failure, and the joy of discovery when things finally worked. Every setback taught us something new, and every small success felt like a victory shared by the whole team.

While we may not have achieved every goal we set out to accomplish, we are proud of the progress we made and the lessons we will carry forward. This project reminded us that science is not only about perfect results, but also about the journey of persistence, teamwork, and creativity. Above all, it showed us the power of collaboration and the excitement of being part of something larger than ourselves.

As we close this chapter, we do so with gratitude, for the guidance we received, for the friendships we strengthened, and for the chance to contribute, in our own way, to addressing the urgent challenge of antimicrobial resistance.

This is not the end, but the beginning of our journey as young scientists.

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