2023/4/15: Consulting information pertaining to Current Genetic Engineering Research
Our team attended an online video conference, consulting knowledge of the field of current genetic engineering research with Prof. Wang. We obtained a general understanding of how some techniques of synthetic biology works, and a comprehensive overview of the general procedure of genetic engineering. Also, we were deeply impressed by the great influence and future potential synthetic biology has.
2023/5/27: Online conference with Mr. Wen for consulting designing and assembly of parts
Our team consulted Dr. Wen for getting a general understanding and inspirational ideas pertaining to designing and assembly of parts. After the conference, we also knew about some of the techniques of connecting DNA sequence and the interesting metaphor that synthetic biology is akin to building blocks. We were ready to start our own fantastic and brand-new journey into synthetic biology.
2023/6/15: Online conference for identifying our research project topic
We held an online conference regarding our team research topic for hours, discussing about the resources and research viewed online pertaining to our topic. We together started our meaningful and valuable topic which is to enable our construct engineered yeast to self-secrete amylase and saccharifying enzyme. The problem we desire to address is that the expensive cost of exogenous enzymes added during alcoholic fermentation. At the same time, we are also trying to introduce sweet potato residue as a raw material of alcoholic fermentation.
2023/6/27: all materials are ready with the help of SubCat and WANG XING.
2023/7/2：Team assembly and Project Kick-Off: Establishing the EcoNzyme Alchemist Team Identity
Our team embarked on four significant activities. First, we engaged in ice-breaking exercises, fostering introductions and facilitating team bonding through interactive games. Next, we held preliminary elections, allowing prospective team leaders to present their speeches and campaign for the role, which served as an important step before finalizing our team leadership. Following this, we collectively undertook a comprehensive review of our project to ensure a common understanding of our goals. To conclude, we together defined our team identity – "EcoNzyme Alchemist", with an inspiring slogan, distinctive visual identity (VI), and self-designed team uniform.
2023/7/3-7/4: Preparation of Luria-Bertani (LB) Solid Medium, Bacterial Culturing, and Plasmid Extraction
Our team successfully prepared the LB and YPD culture media, followed by the inoculation of bacterial strains CCTCC M44055, GA, temA, and Cas-9. Then, we extracted plasmids from strains X-2, XII-5; XI-2, X-3, as well as CCTCC M94055, GA, TemA, and pHCas9, and accurately measured their concentrations.
2023/7/5: Exploring Molecular Biology: A Hands-On Journey through PCR and Gel Electrophoresis
Our team delved into several fundamental molecular biology techniques today. We studied the principles of Polymerase Chain Reaction (PCR) and carried out the process ourselves. Subsequently, we prepared agarose gel and conducted gel electrophoresis. It was a day of both theoretical learning and hands-on experience.
2023/7/6-7/8: Gel Documentation, Gel Extraction, and Over PCR: A Day of Precision and Progress
Our team began by using a gel documentation system to analyze the results of the gel electrophoresis. Following this, we extracted the desired DNA fragments from the gel for further use.
Finally, we embarked on Overlapping PCR (Over PCR), a technique that allows for the fusion of DNA fragments at their overlapping regions. We prepared specific PCR mixtures for four different plasmids. Notably, we successfully fused the GAP promoter, GA gene fragment and CYC1 terminator in one set of reactions, and the TEF1 promoter, GA gene fragment and ADH1 terminator in another. This step was crucial for the assembly of our desired genetic constructs.
2023/7/9: Competent Cell Preparation, DNA Manipulation, and Gel Preparation
We prepared competent yeast cells for transformation, performed restriction digestion and ligation to assemble our desired plasmid, and prepared an agarose gel for electrophoresis. These steps are crucial in our journey towards successful genetic manipulation.
2023/7/10: Preparation and Transformation of Competent Cells
We made significant strides in our project by successfully transforming yeast cells using the lithium acetate method. We began by preparing our competent yeast cells and transformation reagents, then proceeded to introduce our plasmids into the yeast cells. After the transformation, we allowed the cells to recover in a YPD liquid medium. The transformed cells were then spread onto selective agar plates to promote the growth of colonies. In parallel, we prepared a soluble starch agar medium for future experiments. This day marked a pivotal milestone in our project as we successfully incorporated a new genetic material into our yeast cells.
2023/7/11: One Day off
2023/7/12-13: Functional Testing and Solution Preparation
We focused on functional testing of our recombinant yeast cells (GA-temA). We extracted proteins from the cells and analyzed them using SDS-PAGE, which confirmed the successful expression of our target protein. Concurrently, we prepared starch solutions at various pH levels for upcoming enzymatic activity tests. These days were a blend of analytical and preparatory tasks, each crucial for assessing the functionality of our genetic constructs and preparing for subsequent steps in our project.
2023/7/14: Revisiting SDS-PAGE and Initiating Single Clonal Verification
We revisited the SDS-PAGE procedure due to initial results that required further refinement. Our goal was to achieve more precise protein detection. Simultaneously, we initiated the process of single clone verification to confirm the successful gene transformation in yeast. We also prepared an iodine solution, which will be used to confirm successful starch degradation, indicative of the functional expression of our target gene, GA-temA. Despite the need for repetition, this day was a testament to the perseverance and progress inherent in scientific discovery, reminding us that the path to breakthroughs often involves trial and error.
2023/7/15：Gene Expression Confirmation and Enzyme Activity Testing
We made use of the iodine solution we had prepared before. This was used to conduct iodine staining, which confirmed the functional expression of our target gene, GA-temA. We also analyzed the overnight destained SDS-page Protein gel, using a gel documentation system, confirming the successful protein expression. Additionally, we initiated the activity testing of our recombinant enzymes, GA and temA, using the Folin-Wu colorimetric method. This day marked significant progress in our project, as we were able to verify the successful expression and functionality of our target gene.
2023/7/16：Wet lab and Dry lab assembly to discussion about Wiki edition and presentation video
Our team assembled together to distribute work of editing Wiki content, and we also discussed about the framework of presenting our project. To present our project more pithily, we also consulted our supervisor,
and did an efficient mock presentation together to get more familiar with the presentation.
2023/7/17：Mock Presentation and Communicating with audiences for advice
After tidying up our content and modifying our presentation slides, our team presented again in front of some invited audiences with professional knowledge of Synthetic biology. In the latter part of the presentation, we received lots of sincere suggestions from the audiences regarding to our slides design and project introduction, we were really grateful and determined to refine on it further.
2023/7/25 Online meeting for reviewing project and experiment
Our team held a meeting together online, inviting our supervisor together to review the whole project and experiment process together through daily notes we took during the assembly period and the general outline of experiments. Also, we had a Q-and-A section with our supervisor, consulting our confusion and asking for some advice.
2023/7/27 Online meeting regarding work distribution and progress discussion
Our team held an online meeting discussing progress we have made and our future plans for preparing for Wiki and presentation, it was really glad to meet everyone all online again and we did share a meaningful time together distributing work to make our later collaboration more organized and tighter. Furthermore, everyone reported their work progress to keep pace with the project progress.
2023/8/26~28 Yeast fermentation experiment for alcohol production test
After discussion with our instructor, we decided to conduct the alcohol production test to see if our yeast-1974 could successfully produce alcohol. This work lasted 3 days with great help from our instructor Bella and the data was collected for further calculation with the guidance of the kit manual.
2023/8/29 ~ Wiki documentation & construction, Videos editing & submission, et al
Per our work allocation, each one was diligently focused on completing tasks to prepare the final jamboree session and of courses, school classes and homework.
