EHSBNUDate: 1.21 First meeting
Introduce everything about this competition and provide specific information.
Assigned tasks: Started researching for our project topic.
Date: 2.7 Online meeting
Discussed the issue with lab resources. Introduced some lab safety precautions, and assigned some reading/learning tasks to get a general understanding of synthetic biological experiment.
Reading & Learning: Cambell Biology, finish Chapter 3 before the next meeting.
Date: 2.24 Online meeting
Discussed through a series of methods and strategies used in finding laboratory resources, reading papers, and writing literature reviews, etc.
Date: 3.7 Meeting
Discussed about the protein transcription process. Discussed on strategies in finding and reading the articles.
Assigned task: Each team member in the biology department: finish three literature review per person before next Wednesday.
Date: 3.14, 3.21, 3.28
Offline meeting: project brainstorm and discussion.
Presented and discussed through the literature reviews.
Presenter: Lisiqi Ye, Yushan Mei, Zhaoyuan Wu, Gaocheng Zeng, Jiayi Chen, Youzi Wang, Zihe Chen, Yiming Wu, Jingyuan Ma, Yujia Chen, Qingshu Wu.
Organized and collected all accessible topics, assigned reading materials to everyone for further investigation into these topics.
Divide the students into groups, each group focused on one research topic.
Group Hosts: Xirui Wang, Fangming Liu, Yiming Wu, Boyue Deng and Junru Huang, Xinyi Zhao, Yuxuan Wu.
Finally agreed on the topic of limulus blood Factor G proposed by Boyue Deng and Junru Huang.
Date 5.21
Finish the selection about the topic and start designing the experiment: Identify the topic & read some relevant literature
Date 6.10 Online Meeting
Confirm the website construction person in charge of Lisiqi Ye and Zihe Chen Decide not to add new team members
Vote to confirm the approval rate of the leaders of Jiayi Chen and Gaocheng Zeng, and confirm the formal transfer of leadership positions to the Senior One
A meeting will be held within two weeks to design the specific content of the experiment
At present, laboratory resources are limited to the school. Urge all students to find laboratory resources
Date 6.21 Offline Meeting
Build the team wiki.
Design the specific experimental steps.
Complete the project description.
Date 7.9 Offline Meeting
Search for Tachypleus tridentatus clotting Factor G alpha and beta subunit's gene sequence. Perform BLAST and codon optimization. Start designing the PCR primers.
Date 7.12
Design cultural products.
Date 7.25 Offline Meeting:
complete the PCR primers design
Day 9.3 Start the Experiment
Centrifuge the cell pellets: 13,000 rpm for 5 minutes.
Prepare LB medium (100 mL):
1g NaCl
1g tryptone
0.5g yeast extract
1.2-1.5g agar
100 mL water
Sterilization: Autoclave at 121°C for 20 minutes.
Resuspension of cell pellets: Dilute in ddH₂O (1:1000).
Concentration: 100 ng/μL.
Plasmid transfer: 100 μL of competent BL21 (DE3) cells: Add 2 μL of antibiotic, incubate on ice for 15 minutes.
Heat shock: 42°C for 90 seconds.
Recovery: 1 mL of non-antibiotic LB medium, incubate at 37°C for 1 hour.
Centrifuge and plate the cells:
Centrifuge at 4000 rpm for 5 minutes.
Retain 100 μL of supernatant and discard the rest.
Date 9.4
Selected six colonies from the agar plates for both proteins (12 test tubes in total).
Inoculated with 5 mL of LB liquid medium and incubated at 37°C.
Measured OD at 595 nm using a spectrophotometer.
OD is within the appropriate induction range, induced with IPTG at a final concentration of 1 mM.5. Prepared a 1 M stock solution of IPTG, diluted with dd H2O (Before induction, took 400 μL of the culture for pre-induction samples).
Filter-sterilized using a 0.22 μm filter and aliquoted.
Day 9.9
Conducted the SDS-PAGE, the target protein has not been successfully produce
Performed Western Blot for further confirmation:
1. Preparation of transfer buffer: 1 L of transfer buffer consists of 3.55 g of TRIS, 14.4 g of glycine, and 200 mL of methanol, adjusted to a final volume of 1 L with distilled water.
2. Prepare 1 L of TBS (Tris-Buffered Saline) buffer: 2.42g Tris (20mM), 8.76g NaCl (150mM), measured pH=7.45 add 1‰ TWEEN-20 before using.
3. "The Sandwich Structure": Materials needed for Western blot:
A clamp (black cathode at the bottom)
One piece of sponge, pre-soaked in transfer buffer
Three pieces of filter paper, pre-soaked in transfer buffer
The gel
PVDF membrane (requires activation with methanol due to its hydrophobic nature)
Three pieces of filter paper, pre-soaked in transfer buffer
One piece of sponge, pre-soaked in transfer buffer
A clamp (white anode at the top)
Place on ice under a constant current of 300 mA for 2 hours.
4. Blocking: prepare a blocking solution containing 5% (w/v) non-fat dry milk in TBST, seal it and leave it overnight.
Date 9.15
experimental procedureuse on the competent cells:
Add 2 micrograms of plasmid to each of the 3 competent cells and let them stand for 15 minutes
Place the cells at 42 degrees Celsius for 90 seconds
Add 1ml of culture medium and incubate at 37 ° C for 45 minutes
Centrifuge at 4000 rpm for 3 minutes
Coating board
Date 9.22
After the last time the agarose gel electrophoresis did not run out, we replaced the materials by the competent cells. And we did all the process again:
heat shock, resistance screening, OD measurement and IPTG induction expression, SDS-PAGE gel run and harvest.
But this time there were some results, the alpha subunit was sure to express and doing the mass spectrometry on beta submit.
Date 9.29
beta subunit expressed and make preparation before induction,
measure OD and add inducer.
OD is measured as 7.03.
Date 10.2
dilute the material ten times
coating board
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Image 1: Notes example(2024.9.9)