iGEM 2024 — Team nju-china

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Logs

# Logs

Detailed logs for the experiment:

| Date | Works |
| --- | --- |
| 8.5~8.6 | Amplification of the target gene TPS1. |
| 8.7 | Ligate the pYET plasmid containing ADH2p and the TPS1 gene by ligase. Introduce into the receptor E. coli DH5-α for plate coating. |
| 8.8 | Colonies appeared in overnight culture, E. coli monoclonal clones were picked and made into suspension, partially amplified for one day and partially lysed for PCR verification. PCR verification was successful. |
| 8.9 | Use the E. coli suspension amplified for one day, extract the plasmid, carry out enzyme digestion to verify plasmid construction is successful. |
| 8.10 | The plasmid was transferred into the YNB medium of the sensory yeast S288C and cultured for 48 hours for the emergence of single clones. |
| 8.12 | After 4 days of amplification, tryptophan-deficient flat had complete colonies, half of a single clone was picked for PCR verification, and the other half of the single clone that was successfully verified was transferred into 10 ml of pre-culture solution for amplification. |
| 8.16 | After overnight incubation, 500 µl was transferred to 50 ml culture medium for fermentation. Cultures of highly expressed Saccharomyces cerevisiae were carried out and conserved at the end of the four-day culture for subsequent mRNA and resistance assays. |
| 8.19 | Obtaining the vector pYET without TPS1 and promoter. |
| 8.21 | Amplification of promoter PGK1, TEF2, TDH3 from the dried promoter powder obtained from the order. |
| 8.21 | Ligate vector, promoter, TPS gene by ligase. Introduced into receptor E. coli DH5-α and coated plates. |
| 8.22 | E. coli labelled with TPS1 gene using PGK1 promoter did not show any colonies in overnight culture, suspecting that the concentration of plasmid and promoter fragments is not enough, re-PCR promoter PGK1,TEF2,TDH3,TPS gene. Gel electrophoresis was carried out and the gel was cut and recovered. |
| 8.23 | Connect the vector, promoter and TPS gene again by ligase. Introduced into the sensory E. coli DH5-α and coated plates. No colonies appeared in the overnight culture. |
| 8.24 | One day after incubation, colonies appeared on the plate, picked for colony PCR verification, no target band appeared. Recombinant plasmid construction failed. |
| 8.26 | After discussion, we concluded that there was a problem with the plate and reformulated the plate with ampicillin solution. And do PCR promoter PGK1, TET2 again, perform gel electrophoresis test, and cut gel recovery experiments. |
| 8.27 | Ligate vector, promoter, TPS gene by ligase again. Introduced into the receptor E. coli DH5-α and coated plates. Overnight incubation. |
| 8.28 | Tested plate, still no colonies. It is thought that there may be a problem with the ligation. Repeat the ligation. Receptorised E. coli DH5-α was introduced and plates were coated. No colonies appeared in overnight culture. |
| 8.29 | Re-ligated the three promoter genes (PGK1,TEF2,TDH3) with TPS1 and plasmid vector separately, colonies appeared in overnight cultures of yeast using the TEF2,TDH3 promoters, and no colonies appeared in yeast using PGK1. |
| 8.30 | Ligation of TPS1 and plasmid pYET without removal of promoter ADH2p, introduced into sensory E. coli DH5-α. Colonies appeared in overnight culture. |
| 8.31 | Pick E. coli monoclonal clones, make suspension, partially amplify for one day, partially lysed for PCR verification. tEF2, TDH3, two promoter constructs plasmids were successful. |
| 9.1 | Use the E. coli suspension amplified for one day, extract the plasmid, and carry out enzyme digestion to verify that the plasmid constructed with three promoters, TEF2, TDH3, and ADH2P, is successful. |
| 9.2 | The plasmids (TDH3, TEF2, pYET containing ADH2p, and empty pYET) were introduced into the sensory receptor yeast S288C and coated in tryptophan-deficient YNB medium, respectively. |
| 9.5 | After 48 hours of incubation, no colonies appeared in the tryptophan-deficient YNB medium and it was assumed that the YNB had failed, 6.7g/100ml of YNB was reconstituted, and the tryptophan-deficient YNB medium was reconfigured and coated. |
| 9.8 | After 4 days of amplification, the tryptophan-deficient flat had intact colonies, half of a single clone was picked for PCR verification, and the other half of the single clone that was successfully verified was transferred to 10 ml of pre-culture medium for amplification. |
| 9.9 | After overnight incubation of the pre-culture solution, 500 µl was transferred to 50 ml of culture medium for fermentation. Cultures of highly expressed Saccharomyces cerevisiae were carried out and at the end of the four-day culture were conserved for subsequent mRNA and resistance assays. |
| 9.17 | Experiments to compare the growth of different yeast transformants for resistance were carried out using conserved yeast. |
| 9.23 | Use the conserved yeast to extract TOTAL RNA and do RT-qPCR to compare mRNA concentrations and compare different promoter strengths. |

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{% block title %}Engineering{% endblock %}
{% block lead %}Every individual, regardless of background or experience, should have an equal opportunity to engage with scientific knowledge and technological development.{% endblock %}
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![](https://static.igem.wiki/teams/5367/wiki/top-pictures/project-engineering1.png)

Overall Engineering

We Build Prometheus Step by Step

Expert Mode Milestone 1: Fine-Tuning Models for Parts Data Extraction

Expert Mode Milestone 2: Building the Knowledge Graph

Expert Mode Milestone 3: Semantic Search Enhancement

Expert Mode Milestone 4: Plasmid Construction Automation

Freshman Mode Milestone 5

Integration and Cumulative Impact

# The 2024 NJU-CHINA team has developed a large language model of synthetic biology - Prometheus.

In the following section we describe our entire process from the project conception of the model to the success of the build.

## Overall Engineering

![](https://static.igem.wiki/teams/5367/dry/kkk.png)

Our project as a whole went through the DBTL (Design, Build, Test, Learn) cycle.

Design1:

Through interviews and questionnaires with experts, we identified a key challenge in synthetic biology: the lack of a structured knowledge base and organized data. Combining parts and searching for the right parts was difficult and had a high barrier to entry. Therefore, we plan to combine knowledge graphs and large language models to realize the combination of part data, synthetic biology knowledge frameworks and at the same time reduce the threshold of learning and using synthetic biology, so as to promote synthetic biology in a real sense.

Build1:

To address this, we developed a synthetic biology model that processes natural language inputs and outputs part combinations and plasmid designs based on the user's functional requirements.

Test1:

We tested our model by gathering feedback from synthetic biology experts, teachers, and students, as well as individuals with no background in the field. Additionally, we conducted wet lab experiments to validate the model and provide further insights. During this testing, we realized that the current version still had a high learning curve—even for life sciences students—limiting its broader application in advancing synthetic biology.

Learn1&Design2:

This experience inspired us to improve the model by incorporating real-time feedback from users and using experimental data to continuously train and optimize it. As a result, we developed two modes: Expert Mode for users with a deep understanding of synthetic biology, and Freshman Mode, designed for beginners.

Build2:

Freshman Mode allows users to interact through natural language dialogues, with the model performing part searches and plasmid construction in the background, making the tool more accessible and beginner-friendly.

Test2&Learn2:

Freshman Mode was introduced in educational activities, allowing experts, students with a life sciences background, middle school students, and even the public to use the model, and it received widespread praise. It successfully lowered the entry barrier and helped to popularize synthetic biology. With this success, we decided to continue optimizing and iterating the model by reviewing the latest literature and gathering additional feedback through further outreach activities.

# We Build Prometheus Step by Step

## Milestones

We reached several milestones while constructing the Prometheus model:

* 1. We first extracted the part data from the database using fine-tuned Llama3.
* 2. We constructed the part database in the form of a Knowledge Graph.
* 3. We matched the functional descriptions of the parts with the functional requirements of the users using BioSentVec.
* 4. We completed the output of the plasmids.
* 5. We developed Freshman Mode, which is capable of natural language dialogue and automatic extraction of user requirements and we produced a part scoring system and a data feedback system to further prepare for optimizing the model.

Our iGEM project embodies several iterative DBTL (Design, Build, Test, Learn) cycles, where each small loop drives the continuous improvement of our synthetic biology platform. These cycles are connected and form the backbone of our engineering success. Below, we highlight the key DBTL cycles that showcase the progression of our work, each contributing to a larger, interconnected system.

# Expert Mode Milestone 1: Fine-Tuning Models for Parts Data Extraction

Prometheus utilizes the iGEM official database for its part data. However, the extracted metadata often contains excessive text, noise, or truncation issues, making it unsuitable for direct matching with large language models.

To address this, we employed ChatGLM3 and Llama3 with fine-tuning or prompt engineering to extract functional descriptions of parts, condensing them into 2-3 sentences that retain key functional information. This extraction significantly improved the matching success rate of the Sent2Vec model compared to raw data.

Design1 (Dry Lab):

The focus here was on improving the extraction of detailed part functions and environmental context using advanced models like ChatGLM3-8B and Llama3.1.

Build1 (Dry Lab):

We manually annotated 1,000 parts, with two annotators per part. The data was then split into training and testing sets for fine-tuning ChatGLM3. During the training, the loss function converged at around 1.05th epoch. Therefore, we chose to take an early stop at 0.8th epoch based on the loss measurement on the validation set.

Test1 (Dry Lab):

However, ChatGLM3 has a limitation on the maximum number of tokens, which is insufficient for analyzing some iGEM web pages. Other than that, we discovered that ChatGLM3 does not have the ability to precisely extract the function of a biological part – the extraction by which often contains irrelevant information.

Learn1 (HP)&Design2:

We interviewed Prof. Zhen Wu from School of Artificial Intelligence, Nanjing University. He suggested us to apply Llama3, which was the state-of-the-art large language model to perform the extraction.

Build2:

Due to limitation of computational power, we chose to prompt engineer instead of fine-tuning Llama3. The specially designed prompt contains multiple aspects from biological background information to workflow and output format.

Test2:

Llama3 with prompt engineering results in a much better performance than fine-tuning ChatGLM3.

# Expert Mode Milestone 2: Building the Knowledge Graph for Part Organization

The knowledge graph is a method for representing and organizing information through nodes and edges, where nodes represent entities or concepts, and edges denote their relationships. Knowledge graphs are widely used in natural language processing, search engines, and recommendation systems, helping machines understand and reason about information for smarter question-answering and information retrieval. Therefore, we designed a knowledge graph to organize the database of Prometheus.

Design:

Through our pre-project investigation, we identified the need for a more structured and accessible database within iGEM to facilitate efficient part searches. The challenge was that iGEM’s vast data was disorganized and difficult to utilize effectively.

Build:

We developed a knowledge graph using data from the iGEM API and web-scraped content. The knowledge graph organizes synthetic biology parts, categorizing them into distinct types such as promoters, terminators, and operators.

Test:

We ran initial tests using BioSentVec for semantic matching of user input with database parts. The model was able to return relevant parts but struggled with fine-grained distinctions between similar elements. After interviewing Prof. Bo Zhang from the School of Life Science of NJU, we developed an algorithm to assemble other formatted databases with our knowledge graph in order to enrich the biological part that Prometheus can utilize.

Learn:

Feedback showed the need for enhanced precision in part searches. We learned that part categorization and more structured descriptions were necessary to improve the search function.

# Expert Mode Milestone 3: Semantic Search Enhancement for Part Matching

Prometheus allows users to describe parts in natural language and recommends the closest matching parts. Traditional matching algorithms often struggle with varied wording and complex sentence structures, limiting accuracy in real-world applications. To address this, we employed Sent2Vec technology. Given that Prometheus is frequently used in contexts highly relevant to synthetic biology, we specifically utilized the BioSentVec model, retrained on PubMed data.

Design:

After addressing extraction accuracy, we focused on improving semantic search to match user needs with the right parts. A core challenge was inaccurate matches, such as returning terminators when users searched for promoters. We interviewed assistant researcher Zhen Wu and Professor Xinyu Dai from School of Artificial Intelligence, Nanjing University to get more professional advice, such as using word vector technology, Sent2Vec, Word2Vec, and other tools.

Build:

We used BioSentVec to create embeddings for both the part descriptions and user queries. By calculating semantic distances, we aimed to enhance the matching algorithm.

Test:

Initial tests showed that while the system could return relevant parts, mismatches still occurred. We implemented prompt engineering to refine search results, improving the precision of matches.

Learn:

We learned that categorization and embedding calibration were essential. We built a category mapping rule based on previous part classifications, allowing for better matching of parts to user needs.

# Expert Mode Milestone 4: Plasmid Construction Automation

Under the guidance of the HP group, we identified that the model has significant utility for non-experts and beginners. However, their limited background in synthetic biology means that simply providing recommended parts is insufficient for them to create the plasmids they need. Therefore, we require a more comprehensive design for downstream tasks that can directly provide complete plasmids for user reference.

Design:

After communicating with Prof. Shan Chang from the Institute of Bioinformatics and Biomedical Engineering at Jiangsu University of Technology, we found that there was a need for better downstream task design to allow professionals to gain a more convenient experience when designing plasmids. Aiming to streamline the plasmid construction process, we incorporated plasmid backbone selection into the platform. Initially, the system only provided lists of matched parts, but we realized the need for automatic plasmid design.

Build:

We integrated plasmid backbone into our system, allowing automatic assembly of parts based on user-defined criteria. This step improved the platform’s usability and functionality.

Test:

We tested the new system with a variety of users and tasks, discovering that adding more backbone options and auxiliary parts like antibiotic resistance genes further enhanced the design process.

Learn:

This cycle taught us that flexibility in plasmid construction was critical. The inclusion of multiple backbones and auxiliary features allowed users to build more complex, functional plasmids efficiently.

# Freshman Mode Milestone 5: Improving the User Interface with Freshman Mode

After completing the construction of Expert Mode, we validated its adaptability through wet lab experiments and promoted it through HP activities. While it received widespread praise, we also recognized its high barrier to entry. Our team’s original intention is to promote synthetic biology to everyone, enabling all to utilize synthetic biology for their own purposes. The Expert Mode, which is not particularly friendly to newcomers, clearly does not align with the team’s goals. Therefore, we need a more user-friendly interactive method that can be used through common conversational formats in daily life, to lower the threshold for using our model and truly achieve the popularization of synthetic biology. As a result, we packaged it as Expert Mode and developed a more accessible version which is Freshman Mode with conversational capabilities that can automatically extract user requirements. Additionally, we were inspired to optimize the model by collecting data feedback for continuous improvement.

Design:

During the teaching support activities conducted by the HP team, they noticed that the Expert Mode was not suitable for middle and primary school students. Due to their relative lack of foundational knowledge in synthetic biology, they would find it difficult to express their needs for complete synthetic biology products in a structured way, and this frustration could further lead to unnecessary anxiety and resistance toward synthetic biology.

During the wet lab trial, we have demonstrated the utility of the Prometheus model. However, we recognize that effectively using it and inputting the correct requirements still requires some background in synthetic biology. Additionally, we realized that experimental data from Prometheus-guided experiments can be fed back into the model to further enhance its performance. Moreover, we realized that we can score the output parts to optimize Prometheus's part selection process, improving its overall functionality.

Build:

We designed a Freshman Mode by using Llama 3, where the system could guide beginners in natural language, offering step-by-step assistance from part search to plasmid design. This mode was inspired by user feedback from our outreach activities. Freshman Mode can also accept user feedback, experimental data, and user ratings on the model's output parts. We plan to collect sufficient data in the future to further optimize the model.

Test:

We deployed the Freshman Mode on our web platform, allowing non-experts, including middle school students and new researchers, to test it. Feedback showed that the simplified interface significantly lowered the learning curve.

Learn:

Through this cycle, we learned that accessibility was key. The integration of natural language input and a user-friendly interface helped make synthetic biology more approachable, fulfilling our goal of broad promotion. What’s more, we plan to gather sufficient feedback and laboratory data to further optimize Prometheus.

# Integration and Cumulative Impact

Each of these DBTL cycles is interconnected and feeds into the overall evolution of our project. The knowledge graph enables accurate data extraction, which in turn powers semantic search. Freshman Mode lowers the barrier for entry, while automated plasmid construction enhances the system's practical utility for both beginners and experts. Together, these cycles create a robust, user-friendly platform that continually evolves through feedback and learning, driving the successful application of synthetic biology in a broader context.

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{% block title %}Experiments{% endblock %}
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Primer Design

Amplification of Target Gene TPS1

Amplification of Promoters TEF2 and TDH3

Acquisition of Vector pYET without TPS1 and Promoters

Connection and Transformation to Construct Recombinant Expression Vectors

E. coli Plasmid Extraction

Colony PCR Verification

Enzyme Cutting Verification

Yeast Transformation

Measurement of TPS1 mRNA expression

Comparison of resistance growth of yeast transformants expressing TPS1 gene with different promoters

# Primer Design

To amplify the TPS1 gene and the promoters ordered from Genscript (PGK1, TEF2, TDH2) in preparation for constructing a recombinant expression vector for transformation, corresponding homologous arm sequences were designed for the primers. Sequences homologous to the pYET vector and the TPS1 gene were amplified at both ends of the promoter, with additional sequences homologous to the pYET vector added downstream of the TPS1 gene for subsequent ligation.

### 1. TPS1 Amplification Primers, Connected with ADH2p Promoter

- Reverse Primer (R): tcgtgaaggcatgtttaaacTCAGTTTTTGGTGGCAGAGG

### 2. TPS1 Amplification Primers, Connected with TEF2, TDH3, and the Vector Lacking ADH2p Promoter:

-TPS with TDH3 Forward Primer(F):
acttagtttcgaataaacacacataaacaaacaaaATGACTACGGATAACGCTAAGGC

- TPS with TEF2 Forward Primer(F):
cggtcaacgaactataattaactaaacATGACTACGGATAACGCTAAGG

-TPS with Vector Lacking ADH2p Promoter Reverse Primer(R):
tcgtgaaggcatgtttaaacTCAGTTTTTGGTGGCAGAGG

### 3. TDH3 Promoter Amplification Primers:

- TDH3 with Vector Lacking ADH2p Promoter Forward Primer(F):
cggatccatttagcggccgctcagttcgagtttatcattatcaa

- TDH3 with TPS1 Gene Reverse Primer (R):
tttgtttgtttatgtgtgtttattc

### 4. TEF2 Promoter Amplification Primers:

-TEF2 with Vector Lacking ADH2p Promoter Forward Primer(F):
cggatccatttagcggccgcattacccataaggttgtttgt

- TEF2 with TPS1 Gene Reverse Primer (R):
Gtttagttaattatagttcgttgacc

# Amplification of Target Gene TPS1

## 1. Experimental Objectives:

* To amplify the TPS1 gene from yeast S288c.

## 2. Experimental Steps:

1. Pick an appropriate amount of yeast S288c and add 50 µL of 0.1 M NaOH. Let it sit for 10 minutes, then centrifuge and discard the supernatant. Add 50 µL of ddH2O and incubate in a 98°C metal bath for 10 minutes.
2. Prepare a 10 µL PCR system (annealing temperature: 55°C):
   * - ddH2O: 3.3 µL
   * - 2x Phanta Buffer: 5 µL
   * - DMSO: 0.4 µL
   * - dNTP: 0.2 µL
   * - Forward Primer (F): 0.4 µL
   * - Reverse Primer (R): 0.4 µL
   * - Template (yeast): 0.2 µL
   * - Phanta: 0.1 µL
3. Electrophoresis:
   * - Gel Preparation: 1% agarose TAE solution. Dissolve 4 g of agarose in 200 mL TAE by heating, then add 4 µL of nucleic acid dye after cooling slightly.
   * - Electrophoresis: Marker: DL8000; run at 180 V for 20 minutes.
4. Gel Recovery:
   * - Cut out the target band from the gel and place it in a clean centrifuge tube, then weigh it.
   * - Add an equal volume of V1 gel solution to the tube, incubate at 55°C for 10 minutes, inverting 2-3 times to ensure complete melting.
   * - Briefly centrifuge to collect the liquid from the tube walls.
   * - Place the adsorption column in a collection tube, add the gel solution to the column, and centrifuge at 12,000 rpm for 1 minute.
   * - Add 500 µL of wash solution W2 (with anhydrous ethanol) to the column, centrifuge at 12,000 rpm for 2 minutes.
   * - Repeat the previous step.
   * - After a final spin at 12,000 rpm for 2 minutes, place the column in a clean centrifuge tube, open the lid, and let it dry for 5 minutes.
   * - Add 5 µL of TE to the adsorption membrane, let it sit at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute to collect the target gene and measure its concentration.

## 3. Experimental Results:

Successfully amplified the TPS1 gene.

![](https://static.igem.wiki/teams/5367/wet/1-tps.jpg)

TPS1 PCR Electrophoresis Validation Image

# Amplification of Promoters TEF2 and TDH3

## 1. Experimental Objectives:

* To amplify the TEF2 and TDH3 promoters.

## 2. Experimental Steps:

1. Prepare a 10 µL PCR system (annealing temperature: 51-55°C):
   * - ddH2O: 3.3 µL
   * - 2x Phanta Buffer: 5 µL
   * - DMSO: 0.4 µL
   * - dNTP: 0.2 µL
   * - Forward Primer (F): 0.4 µL
   * - Reverse Primer (R): 0.4 µL
   * - Template (pYET): 0.2 µL
   * - Phanta: 0.1 µL
2. Gel Electrophoresis:
   * - Gel Preparation: 1% agarose TAE solution. Dissolve 4 g of agarose in 200 mL TAE by heating, then add 4 µL of nucleic acid dye after cooling slightly.
   * - Electrophoresis: Marker: DL8000; run at 180 V for 20 minutes.
3. Gel Recovery:
   * - Cut out the target band from the gel and place it in a clean centrifuge tube, then weigh it.
   * - Add an equal volume of V1 gel solution to the tube, incubate at 55°C for 10 minutes, inverting 2-3 times to ensure complete melting.
   * - Briefly centrifuge to collect the liquid from the tube walls.
   * - Place the adsorption column in a collection tube, add the gel solution to the column, and centrifuge at 12,000 rpm for 1 minute.
   * - Add 500 µL of wash solution W2 (with anhydrous ethanol) to the column, centrifuge at 12,000 rpm for 2 minutes.
   * - Repeat the previous step.
   * - After a final spin at 12,000 rpm for 2 minutes, place the column in a clean centrifuge tube, open the lid, and let it dry for 5 minutes.
   * - Add 5 µL of TE to the adsorption membrane, let it sit at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute to collect the target gene and measure its concentration.

## 3. Experimental Results:

Successfully amplified the TDH3 and TEF2 promoters.

![](https://static.igem.wiki/teams/5367/wet/2-tdh3-tef2-pcr.jpg)

TDH3 TEF2 PCR Electrophoresis Validation Image

# Acquisition of Vector pYET without TPS1 and Promoters

## 1. Experimental Objectives:

* To obtain a pYET plasmid with the original ADH2p promoter removed, using an existing pYET that has the target gene excised, in preparation for subsequent connections with TDH3 and TEF2.

## 2. Experimental Steps:

1. Prepare a 50 µL restriction enzyme system at 37°C for 2 hours:
   * - Amplified pYET: 10 µL
   * - NotI: 5 µL
   * - MssI: 5 µL
   * - 10x Buffer: 5 µL
   * - ddH2O: 25 µL
2. Gel Electrophoresis:
   * - Gel Preparation: 1% agarose TAE solution. Dissolve 4 g of agarose in 200 mL TAE by heating, then add 4 µL of nucleic acid dye after cooling slightly.
   * - Electrophoresis: Marker: DL8000; run at 180 V for 20 minutes.
3. Gel Recovery:
   * - Cut out the target band from the gel and place it in a clean centrifuge tube, then weigh it.
   * - Add an equal volume of V1 gel solution to the tube, incubate at 55°C for 10 minutes, inverting 2-3 times to ensure complete melting.
   * - Briefly centrifuge to collect the liquid from the tube walls.
   * - Place the adsorption column in a collection tube, add the gel solution to the column, and centrifuge at 12,000 rpm for 1 minute.
   * - Add 500 µL of wash solution W2 (with anhydrous ethanol) to the column, centrifuge at 12,000 rpm for 2 minutes.
   * - Repeat the previous step.
   * - After a final spin at 12,000 rpm for 2 minutes, place the column in a clean centrifuge tube, open the lid, and let it dry for 5 minutes.
   * - Add 5 µL of TE to the adsorption membrane, let it sit at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute to collect the target gene and measure its concentration.

## 3. Experimental Results:

Successful enzyme digestion.

![](https://static.igem.wiki/teams/5367/wet/3-pyet-noti-mssi.jpg)

pYET Enzyme Digestion Electrophoresis Validation Image

# Connection and Transformation to Construct Recombinant Expression Vectors

## 1.Experimental Objectives

* To connect TEF2, TDH3 with the pYET vector lacking the ADH2p promoter and the TPS1 gene.
* To connect the pYET vector retaining the ADH2p promoter with the TPS1 gene.
* To transform into E. coli and select for ampicillin-resistant single colonies.

## 2.Experimental Steps

### 1. Connection

#### - Multi-fragment Connection (TEF2 or TDH3, pYET lacking the ADH2p promoter, and TPS1 gene)

* - For the multi-fragment recombination reaction:
  + - pYET vector: 0.02 × number of base pairs in the cloning vector (ng)
  + - TPS1: 0.02 × number of base pairs in the TPS1 gene
  + - Promoter: 0.02 × number of base pairs in the promoter
* - 10 µl Connection System
  + - pYET vector: 1 µl
  + - TPS1: 2 µl
  + - Promoter: 2 µl
  + - ddH2O: 5 µl
  + - Mix gently, incubate at 50°C for 15 min, then immediately place on ice.

#### - Single-fragment Connection (retaining the ADH2p promoter on pYET)

* - For the single-fragment recombination reaction:
  + - pYET vector: 0.02 × number of base pairs in the cloning vector (ng)
  + - TPS1: 0.04 × number of base pairs in the TPS1 gene
* - 10 µl Connection System
  + - pYET vector: 1 µl
  + - TPS1: 2 µl
  + - Promoter: 2 µl
  + - ddH2O: 5 µl
  + - Mix gently, incubate at 50°C for 15 min, then immediately place on ice.

### 2. Transformation

* - Place the E. coli DH5α competent cells on ice to thaw.
* - Add 10 µl of the recombinant product to 50 µl of competent cells, gently mix by flicking the tube, and incubate on ice for 30 min.
* - Heat shock at 42°C for 30 s, then immediately place on ice for 3 min.
* - Add 500 µl of LB liquid medium (without antibiotic) and incubate at 37°C for 1 h with shaking.
* - Centrifuge at 5000 rpm for 5 min, discard most of the supernatant, and plate the remaining supernatant on LB medium with ampicillin, and culture overnight.

Successful enzyme digestion.

![](https://static.igem.wiki/teams/5367/wet/4-dh5-alpha-tdh3-tef2.jpg)

Screening of E.coli transformed with TPS1

# E. coli Plasmid Extraction

## 1. Experimental Objectives

* To obtain amplified plasmids through E. coli culture.
* To acquire relatively pure target plasmids.
* To conduct PCR verification of the target plasmids.

## 2. Experimental Steps

1. Check Equipment:
   * - Confirm that RNase A has been added to Buffer P1.
   * - Verify that anhydrous ethanol is present in the Wash Solution.
   * - Check for precipitation in Buffers P2 and P3.
2. Cell Collection:
   * - Take 1.5-5 mL of overnight cultured bacterial liquid, centrifuge at 8,000 × g for 2 minutes to collect cells, and discard the supernatant.
3. Lysis:
   * - Add 250 μl of Buffer P1 to the pellet and resuspend thoroughly.
   * - Add 250 μl of Buffer P2, gently invert the tube 5-10 times to mix. Incubate at room temperature for 2-4 minutes.
4. Centrifuge:
   * - Add 350 μl of Buffer P3, gently invert the tube 5-10 times to mix.
5. Collect Supernatant:
   * - Centrifuge at 12,000 × g for 5-10 minutes. Transfer the supernatant to a binding column, and centrifuge at 8,000 × g for 30 seconds; discard the collection.
6. Washing:
   * - Add 500 μl of Wash Solution, centrifuge at 9,000 × g for 30 seconds, discard the liquid, and repeat once.
   * - Centrifuge the empty binding column at 9,000 × g for 1 minute.
7. Elution:
   * - Place the binding column into a clean 1.5 mL centrifuge tube, add 50-100 μl of Elution Buffer to the center of the membrane, let sit at room temperature for 1 minute, then centrifuge for 1 minute. Save the DNA solution in the tube.
8. Construct PCR System for Plasmid Verification

# Colony PCR Verification

## 1. Experimental Objective:

* To confirm the successful insertion of the TPS1 gene and promoters TEF2, TDH3 into the pYET plasmid.

## 2. Experimental Procedure:

1. Take 3 positive clones, add 10 μl of sterile water, and mix.
2. Use 1 μl of the bacterial liquid as PCR template. Add the remaining liquid to 500 μl of LB(Amp) medium and shake at 37°C for 5 hours.
3. 10 μl PCR System:
   * - ddH2O: 3.2 μl
   * - Taq enzyme: 5 μl
   * - DMSO: 0.4 μl
   * - Primer F: 0.2 μl
   * - Primer R: 0.2 μl
   * - Template (bacterial liquid): 1 μl
   * - Annealing temperature: 56°C
4. Perform agarose gel electrophoresis.
5. Select the culture liquid containing the correct positive clone and add to 3 mL of LB(Amp) medium, shaking for 5 hours.

## 3. Experimental Results:

PCR validation shows TDH3, TEF2 are ligated to pYET plasmid.

Successful enzyme digestion.

![](https://static.igem.wiki/teams/5367/wet/5-dh5-alpha-tdh3-tef2-pcr.png)

DH5-alpha TDH3 TEF2 TPS1 PCR Electrophoresis Validation Image

PCR validation shows TPS1 ligated to pYET plasmid without removal of the ADH2p promoter

Successful enzyme digestion.

![](https://static.igem.wiki/teams/5367/wet/6-dh5-alpha-adh2p-tps1.jpg)

DH5-alpha ADH2 TPS1 PCR Electrophoresis Validation Image

# Enzyme Cutting Verification

## 1. Experimental Objective:

* To extract plasmids from the correct positive clones.

## 2. Experimental Steps:

1. Check that RNase A has been added to Buffer P1; verify that anhydrous ethanol is present in the Wash Solution; check for precipitation in Buffers P2 and P3.
2. Take 3 mL of bacterial liquid, centrifuge at 8,000 × g for 2 minutes to collect cells, and discard the supernatant.
3. Add 250 μl of Buffer P1 to the pellet, resuspend thoroughly.
4. Add 250 μl of Buffer P2, gently invert the tube 5-10 times to mix. Incubate at room temperature for 2-4 minutes.
5. Add 350 μl of Buffer P3, gently invert the tube 5-10 times to mix.
6. Centrifuge at 12,000 × g for 5-10 minutes. Transfer the supernatant to a binding column, centrifuge at 8,000 × g for 30 seconds; discard the collection.
7. Add 500 μl of Wash Solution, centrifuge at 9,000 × g for 30 seconds; discard the collection.
8. Repeat the last step once.
9. Centrifuge the empty binding column at 9,000 × g for 1 minute.
10. Place the binding column into a clean 1.5 mL centrifuge tube, add 30 μl of Elution Buffer to the center of the membrane, let sit for 1 minute, then centrifuge for 1 minute. Save the DNA solution in the tube and measure the concentration.

### Enzyme Cutting Gel Verification:

* 10 μl Enzyme Cutting System:
  + - Plasmid: 8 μl
  + - HindIII: 0.5 μl
  + - BglII: 0.5 μl
  + - 10× Green Buffer: 1 μl
* Incubate in a 37°C water bath for 0.5 hours, followed by gel electrophoresis.

## 3. Experimental Results:

Enzyme digestion validation shows TDH3, TEF2 ligated to pYET plasmid.

![](https://static.igem.wiki/teams/5367/wet/7-dh5-alpha-tdh3-tef2-digestion.jpg)

DH5-alpha TDH3 TEF2 TPS1 Enzyme Digestion Electrophoresis Validation Image

Enzyme digestion validation shows TPS1 ligated to pYET plasmid without removal of the ADH2p promoter.

Enzyme digestion validation shows TDH3, TEF2 ligated to pYET plasmid.

![](https://static.igem.wiki/teams/5367/wet/8-dh5-alpha-adh2p-digestion.jpg)

DH5-alpha ADH2-TPS1 Enzyme Digestion Electrophoresis Validation Image

# Yeast Transformation

## 1. Experimental Objectives

* To introduce the target plasmid into competent yeast cells.
* To obtain individual yeast colonies containing the target plasmid.

## 2. Experimental Steps

1. Prepare the pre-mix solution. For each plasmid transformation (one reaction), 360 μL of pre-mix solution is required:
   * - Solution C: 350 μl
   * - Plasmid (approximately 200 ng/μl): 5 μl
   * - ddH2O: 5 μl
2. Pipette 360 μL of the pre-mix solution into the competent cells, and repeatedly pipette to thoroughly suspend the yeast cells at the bottom of the centrifuge tube in the pre-mix solution.
3. Place in a 30°C water bath for heat shock for 45-60 minutes, mixing every 10 minutes. After the water bath, centrifuge at 3,000 g for 3 minutes.
4. Discard the supernatant and resuspend the pellet in 0.5 mL of YPD Plus, then incubate on a shaking incubator at 30°C for 30-60 minutes. Centrifuge at 3,000 g for 5 minutes and discard the supernatant.
5. Add 1 mL of sterile water or 0.9% NaCl to resuspend the pellet. Dilute separately to 10-fold and 100-fold, and plate onto the appropriate selective media.
6. Incubate at a constant temperature of 30°C for 3-5 days until yeast colonies appear on the plates.

## Precautions:

* All steps of the transformation should be conducted under sterile conditions. YNB Plus should be stored at -20°C after opening to prevent contamination.
* To ensure transformation efficiency, competent cells should not be directly frozen in liquid nitrogen.
* Increasing the quality of the yeast plasmid can improve transformation efficiency.

## 3. Experimental Results:

Yeast monoclonal to pYET plasmid containing TDH3,TEF2 promoter

![](https://static.igem.wiki/teams/5367/wet/9-s288c-pyet-tdh3-tef2.jpg)

S288c TDH3 TEF2 pYET Cultivation Image

Yeast monoclonal to pYET plasmid containing ADH promoter

![](https://static.igem.wiki/teams/5367/wet/10-s288c-adh2p.jpg)

S288c ADH2 Cultivation Image

Half of the yeast monoclonal clone was picked for PCR verification, and the other half was successfully verified for fermentation culture. The results of PCR verification proved that the plasmids in the yeast monoclonal clone contained TDH3, TEF2 and ADH promoters.

![](https://static.igem.wiki/teams/5367/wet/11-s288c-pcr.png)

S288c TDH3 TEF2 TPS1 PCR Electrophoresis Validation Image

![](https://static.igem.wiki/teams/5367/wet/12-s288c-adh-pcr.jpg)

S288c ADH2 pYET PCR Electrophoresis Validation Image

# Measurement of TPS1 mRNA expression

## 1. Purpose of the experiment

* Compare the mRNA intensity using TDH3, TEF2, ADH2p, and blank groups
* To demonstrate overexpression of the TPS1 gene.
* To demonstrate the utility of the Prometheus model recommended promoters TDH3 and TEF2.

## 2. Experimental Procedures

### Total RNA extraction

1. Add 200ul of PBS to the yeast precipitate and resuspend, take 50ul of RNA extraction.
2. Add 950ul Trizol, shake vigorously and place on ice.
3. Add 200ul chloroform, shake vigorously and mix well, let it stand at room temperature for 10min, centrifuge at 16000rpm for 20min (4℃).
4. Pipette the supernatant 500ul into a new centrifuge tube, add equal volume of isopropanol, shake and let it stand at -20℃ for 1h.
5. Centrifuge at 16000rpm for 20min (4℃) and discard the supernatant.
6. Configure 75% ethanol (DEPC water: anhydrous ethanol = 1:3), take 800ul into the centrifuge tube, turn up and down.
7. 12000rpm centrifugation for 20min (4℃), discard the supernatant, dry.
8. Add 20ul DEPC water, mix well and measure the concentration.

### Reverse transcription

1. Genomic RNA removal
   * RNase-free centrifuge tube is configured with the following mixture, then blow and mix well, 42 ℃ for 2min.
   * RNase-free ddH2O 10ul
   * 4x gDNA wiper Mix 4ul
   * Template RNA 2ul
2. Preparation of reverse transcription reaction system
   * Add 4ul of 5x HiScript III qRT SuperMix to the reaction solution in the first step, blow and mix well.
3. Perform reverse transcription reaction
   * 37℃ 15min
   * 85℃ 5sec

### qPCR

1. Prepare 20ul of system in a qPCR tube.
   * ddH2O 7.2ul
   * 2x ChamQ SYBR qPCR Master Mix (Without ROX) 10ul
   * cDNA 2ul
   * Primer F 0.4ul
   * Primer R 0.4ul
2. qPCR was performed under the following conditions
   * Pre-denaturation Rep:1 95℃ 30s
   * Cycle Rep:40 95℃ 10s ; 60℃ 30s
   * Melting curve Rep:1 95℃ 15s ; 60℃ 60s; 95℃ 15s

## 3. Experimental Results:

The mRNA concentrations of the three promoters TDH3, TEF2, and ADH2 expressing TPS1 were determined using fluorescence quantitative PCR, and the relative expression of mRNAs expressing TPS1 from the four promoters was compared by Ct values as a means of comparing the relative strengths of the expression of the three promoters, as well as the elevation of the mRNA concentration with respect to the blank group.

Each promoter included a blank group, we used two yeast monoclones that were successfully transferred into the promoter for fermentation, and after three days 1ml was removed and centrifuged at 4000g at 4°C to obtain the bacteriophage for mRNA extraction, reverse transcription, and fluorescent quantitative PCR.

Amplification curves were plotted and melting curves were determined based on Ct values.

| Color | Position | Sample Name | Gene Name | Condition Name | Cq | Cq Mean | Cq Error | Excluded | Sample Type | Sample Type RQ | Gene Type | Condition Type | Replicate Group | Ratio | Ratio Error | Normalized Ratio | Normalized Ratio Error | Scaled Ratio | Scaled Ratio Error | Dye | Edited Call | Failure | Slope | EPF | Notes | Sample Prep Notes | Number |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 255;132;197;238 | A1 | Sample 1 | ADH2-1 |  | 19.7 | 19.7 | 0 | Unchecked | Unknown | Unknown | Target | Unknown |  | - | - | - | - | - | - | SYBR Green I |  | No ratio because no reference gene defined | 0.51 | 2.98 |  |  | 1 |
| 255;103;103;103 | A2 | Sample 2 | pYET-1 |  | 31.84 | 31.84 | 0 | Unchecked | Unknown | Unknown | Target | Unknown |  | - | - | - | - | - | - | SYBR Green I |  | No ratio because no reference gene defined | 0.79 | 3.92 |  |  | 2 |
| 255;242;138;21 | A3 | Sample 3 | ADH2-2 |  | 21.28 | 21.28 | 0 | Unchecked | Unknown | Unknown | Target | Unknown |  | - | - | - | - | - | - | SYBR Green I |  | No ratio because no reference gene defined | 0.9 | 4.3 |  |  | 3 |
| 255;250;215;15 | A4 | Sample 4 | pYET-2 |  | 31.12 | 31.12 | 0 | Unchecked | Unknown | Unknown | Target | Unknown |  | - | - | - | - | - | - | SYBR Green I |  | No ratio because no reference gene defined | 0.7 | 3.69 |  |  | 4 |
| 255;103;154;47 | A5 | Sample 5 | TEF2-1 |  | - | - | 0 | Unchecked | Unknown | Unknown | Target | Unknown |  | 0.00E+00 | - | - | - | - | - | SYBR Green I |  | No ratio because no reference gene defined | 0.21 | 2.5 |  |  | 5 |
| 255;180;73;149 | A6 | Sample 6 | TEF2-2 |  | 23.32 | 23.32 | 0 | Unchecked | Unknown | Unknown | Target | Unknown |  | - | - | - | - | - | - | SYBR Green I |  | No ratio because no reference gene defined | 0.72 | 3.72 |  |  | 6 |
| 255;95;103;174 | A7 | Sample 7 | TDH3-1 |  | 19.51 | 19.51 | 0 | Unchecked | Unknown | Unknown | Target | Unknown |  | - | - | - | - | - | - | SYBR Green I |  | No ratio because no reference gene defined | 0.66 | 3.52 |  |  | 7 |
| 255;205;18;55 | A8 | Sample 8 | TDH3-2 |  | 20.33 | 20.33 | 0 | Unchecked | Unknown | Unknown | Target | Unknown |  | - | - | - | - | - | - | SYBR Green I |  | No ratio because no reference gene defined | 0.62 | 3.37 |  |  | 8 |

Fluorescent Quantitative PCR Data Table

![](https://static.igem.wiki/teams/5367/wet/14amplification-curve.jpg)

Amplification curve of TDH3, TEF2, ADH2 and pYET

Legend: Light blue: ADH2-1; Gray: pYET-1; Orange: ADH2-2; Yellow: pYET-2; Green: TEF2-1; Pink: TEF2-2; Dark blue: TDH3-1; Red: TDH3-2.

![](https://static.igem.wiki/teams/5367/wet/15melting-curve.jpg)

Dissolution Curve of TDH3, TEF2, ADH2 and pYET

Legend: Light blue: ADH2-1; Gray: pYET-1; Orange: ADH2-2; Yellow: pYET-2; Green: TEF2-1; Pink: TEF2-2; Dark blue: TDH3-1; Red: TDH3-2.

From the amplification curves, it can be seen that all six experimental groups of the three promoters greatly increased the expression of TPS1, and the Ct values were all in the range of 20-25. The Ct values of the blank group were all after 30. The successful expression of the promoters TDH3 and TEF2 provided by Prometheus was demonstrated, proving the usefulness of the model. Comparing the expression of TDH3 promoter and the original ADH2 of pYET plasmid, the TDH3 promoter increased the mRNA concentration of TPS1 by nearly 50% compared to ADH2, proving once again the superiority of Prometheus in component selection.

* **Relative expression of TDH3 relative to blank group:** 2(31.48 - 19.92) = 3019
* **Relative expression of TEF2 relative to blank group:** 2(31.48 - 23.32) = 286
* **Relative expression of ADH2 relative to blank group:** 2(31.48 - 20.49) = 2033
* **Relative expression of TDH3 relative to ADH2 group:** 2(20.49 - 19.92) = 1.484

![](https://static.igem.wiki/teams/5367/wet/relative-expression-to-blank-group.png)

Relative expression to blank group

# Comparison of Resistance Growth of Yeast Transformants Expressing TPS1 Gene with Different Promoters

## 1. Experimental Objectives

* To verify that alginate is resistant to yeast growth.
* To investigate whether there is a difference in resistance between different promoters expressing alginate.

## 2. Experimental Procedures

1. In the aseptic operating table, take 500μl of bacterial solution from the seed vials of two experimental groups with different promoters and add it into 50ml of culture medium, incubate at 37°C and 220rpm for 40h for amplification and fermentation.
2. Every 2h interval, take 1ml culture solution and add it to the cuvette, measure the OD value of the bacterial solution.

### Precautions:

* Aseptic operation is required throughout the process, and all culture conditions are strictly controlled to be consistent.
* Set up experimental group (TEF2, TDH3, ADH2), negative control group (pYET), blank control group (blank culture solution added without sterile solution).

## 3. Experimental Results

By plotting the time and OD curves, the yeast with different promoters showed a better growth trend at thirty-seven degrees Celsius than the blank group.

![](https://static.igem.wiki/teams/5367/wet/7igem-od.png)

OD value versus time curve under cultivation at 37°C.

| Type | OD1 | OD2 |
| --- | --- | --- |
| blank | 0.009 | 0.011 |
| blank | 0.001 | 0.001 |
| blank | 0.001 | 0.002 |
| blank | 0.001 | 0.002 |
| blank | 0 | 0 |
| blank | 0.001 | 0 |
| blank | 0.002 | 0.003 |
| blank | 0.001 | -0.001 |
| blank | 0.001 | 0.001 |
| blank | 0.002 | 0.001 |
| blank | 0 | 0 |
| blank | 0 | -0.001 |
| TEF | -0.025 | -0.017 |
| TEF | 0.006 | 0.010 |
| TEF | 0.112 | 0.103 |
| TEF | 0.424 | 0.417 |
| TEF | 1.329 | 1.325 |
| TEF | 2.088 | 2.076 |
| TEF | 2.605 | 2.611 |
| TEF | 2.830 | 2.882 |
| TEF | 2.900 | 2.921 |
| TEF | 3.113 | 3.115 |
| TEF | 3.232 | 3.241 |
| TEF | 3.613 | 3.624 |
| TDH | -0.08 | -0.148 |
| TDH | 0.046 | 0.052 |
| TDH | 0.18 | 0.169 |
| TDH | 0.263 | 0.256 |
| TDH | 0.443 | 0.451 |
| TDH | 0.854 | 0.836 |
| TDH | 1.867 | 1.882 |
| TDH | 2.212 | 2.206 |
| TDH | 2.566 | 2.579 |
| TDH | 2.682 | 2.604 |
| TDH | 2.791 | 2.803 |
| TDH | 3.347 | 3.339 |
| ADH | -0.029 | -0.041 |
| ADH | -0.003 | -0.006 |
| ADH | 0.046 | 0.05 |
| ADH | 0.115 | 0.128 |
| ADH | 0.207 | 0.194 |
| ADH | 0.317 | 0.332 |
| ADH | 0.815 | 0.821 |
| ADH | 1.329 | 1.335 |
| ADH | 1.854 | 2.178 |
| ADH | 2.011 | 2.017 |
| ADH | 2.466 | 2.441 |
| ADH | 3.219 | 3.21 |
| pYET | -0.017 | -0.031 |
| pYET | -0.011 | -0.003 |
| pYET | 0.019 | 0.034 |
| pYET | 0.056 | 0.072 |
| pYET | 0.079 | 0.081 |
| pYET | 0.139 | 0.143 |
| pYET | 0.170 | 0.175 |
| pYET | 0.330 | 0.351 |
| pYET | 0.792 | 0.796 |
| pYET | 1.329 | 1.312 |
| pYET | 1.725 | 1.686 |
| pYET | 2.466 | 2.475 |

OD600 over Time Data Table

![](https://static.igem.wiki/teams/5367/wet/17tolerane-test.jpg)

Photos of cultivation at 37°C.

Legend: From left to right: TDH3, TEF2, ADH2, pYET.

The above figure shows the shaker flasks incubated for 12 h. From left to right are TDH3, TEF2, pYET, and ADH2. a clear difference in the resilience of the blank group and the yeasts that achieved TPS1 overexpression to the unsuitable temperature can be seen.

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