We participated in the determination and feasibility analysis of the overall plan from an experimental perspective.
We have finalized the experimental plan.
We reviewed the laboratory safety knowledge we had previously learned and conducted safety training specifically for our experiments.
We learned how to use the relevant instruments.

We learned and conducted HEK293T cell passage experiments for the first time.
We learned and conducted HEK293T cell transfection experiments for the first time, and a cell exchange was performed.
We conducted HEK293T cell passage experiments, and observe the results of cell transfection.
Results:30% cell floated.
Possible reason:while transfecting plasmid into cell culture dish, the cell was blown up. Observed the result of cell passage experiments.
Results:The cells are in a bad condition, 10% growth rate. possible reason:There is no enough water in the incubator.
Under the guidance of our teacher, we learned how to construct plasmids on snapgene software.

Amplifed and extracted plasmids:
2401 pGIP-luc
2402 pGIP-GI-Gal4 wlmiBS
2408 miR-2BS
2409 9xUAS-luc
2410 miR-1
2411 sponge
Results:We got 2401 370uL 127.7ug/uL  2409 180uL 88.9ug/uL
2402 90uL 229.3ug/uL   2410 370uL 68.8ug/uL
2408 370uL 129.1ug/uL  2411 180uL 67.5ug/uL Resuscitat the last frozen HEK293T cells.
Take 500uL of cells and add them to a large dish to allow them to reproduce for 2 days.
After 2 days, when observing the cells, we found that they are full. After digestion and resuspension, 300uL of cells were taken and divided into 2 large plates for passage, one for cryopreservation and one for subsequent experiments.

We prepared LB culture medium and prepared ampicillin sodium antibacterial solution.
Mix and plate to make 88 ampicillin sodium media. Supplemented with laboratory pipette tips.
Amplify and extract plasmids:
2403 pcDNA Lov-VP64
2412 pcDNA GI-Gal4
Renilla
pcDNA3.1
We passaged HEK293T cells. We freezed part of this batch of cells and continued to freeze the other part too observe the growth.
Because the cells are filled with the large dish. So We did the cell passag, passed 200uL cells into a large dish and 100uL cells into a medium dish. After passage, the cell density was reduced and they get better environment to live.

We used high-fidelity PCR technology to obtain the linear fragments that I needed from three plasmids. We searched online for the instruments of the high-fidelity enzyme to determine the system quantity, and then set up the program for the PCR instrument.
1. We used primer pGV-F and pGV-R to get pcDNA3.1 fragment from plasmid pcDNA3.1- Gal4-VP64 (inverse PCR)
2. We used primer pGD-F and pGD-R to get pGIP(1) fragment from plasmid pGIP-GI-Gal4- DBD
3. We used primer pGL-F and pGL-R to get pGIP(1) fragment from pGIP-LOV-VP16 the system: pcDNA3.1   pGIP(1)   pGIP(2)
mix             25       25     25
F and R      2         2        2
(DNA)        1          1         2
ddH2O       20      20      20

The three tubes of PCR products were lost due to unknown reasons. These are the products we made yesterday. Today I used pGG-R and pGG-F to get pGIP(3); DG-F, GD-R to get GI-Gal4-DBD; LV-F, LV-R to get LOV-VP16. We configured 400mL of LB medium.

Amplified and extracted plamids:
Flag-PKR2
Pcaggs
We got Flag-PKR2 100uL(50uL 92.1ng/uL, 50uL 71.0ng/uL) and Pcaggs 180uL(90uL 38.9ng/uL, 90uL 42.8ng/uL)
We used GV-F,GV-R to get GV-2ER(1); GV-F, GV-R2 to ge GV-2ER(2),through PCR.
We also remade the product we lost yesterday.
We used pGV-F and pGV-R to get pcDNA3.1.
We used pGD-F and pGD-R to get pGIP(GI).
We used pGL-F and pGL-R to get pGIP(LOV).

We followed the senior apprentices to learn the part of measuring luciferase activity in the immunofluorescence experiment, which laid the groundwork for the detection of protein expression int the future.

Due to the shortage of water in incubator, we had a poor cell status. So we resuscitated a tube of cells, and cultured them in two cultures, one for later experiments and one for preservation.

We also carried out agarose gel electrophoresis experiments.

The cells were in good condition and dense, so cell passage and cell planking were performed to prepare for cell transfection.

The cell density is about 50%-60% and we did the cell transfection.
Six hours after transfection, a culture-medium exchange was performed, replacing the sugar- free medium with glucose gradient medium.
We did the fluorescence detection of the fluorescence report experiment, and the following are the results of the experiments.
We did PCR experiments and amplify and extract plasmids(2401 pGIP-Luc and pcDNA 3.1).
We conducted gel electrophoresis experiment and learned single clone operations.

We tried a new plasmid template, pcDNA3.1-Gal4-VP64-2ER, to finish this PCR. pGV-F and PGV-R were used to perform reverse PCR on this template.
The obtained products were combined with PGIP-related PCR products obtained on April 21 for agarose gel electrophoresis.
We got the expected strip and planned to cut and recycle it the next day.
We recycled plasmid: pcDNA3.1, pGIP GV-2ER, pGIP Gal4, pGIP LOV-VP16.
We amplified and extract plasmids: pcDNA3.1-Gal4-VP16-2ER, pcDNA3.1, renila.

The pGIP luc template was amplified by primestar enzyme, and the amplification results were verified by gel electrophoresis experiment.
Conduct elution experiments to increase concentration.
Recycled products (positive stranded DNA, GV-2ER，DBD，LOV-VP16），and conducted concentration measurements. We Stored them at -20 degrees Celsius.

Due to unsatisfactory enzyme digestion results, the new plasmid was transformed and sequenced.
We conducted monoclonal experiments (pGIP luc, Renila et al.) and recovered DNA.

We conducted gradient PCR experiments and explored and discussed the optimal experimental temperature. The final temperature was determined to be 64 degrees Celsius.

We conducted plasmid extraction experiments (pGIP luc, pGIP luc, renila) and carried out recovery, recombination, and transformation experiments.
Perform multiple PCR experiments using the previously identified optimal temperature (64 degrees Celsius).

After transforming the plasmid, coat the plate and observe the colony.
We conducted amplification experiments on plasmids.
We organized all the samples in the refrigerator, conducted activity tests on all the samples, and cleaned 7 tubes of inactive samrples.

We observed the conversion results and found the presence of mixed bacteria. The speculated reason is that the recombinant system was not completely mixed, the DNA fragments were somewhat degraded, and there were problems with the original pGIP plasmid.
We collected fresh DNA, specifically VP64 (p), pGIP (VP64), DBD, pGIP (DBD), VP16, pGIP (VP16).
We conducted plasmid recombination experiments and conducted transformation experiments after thorough mixing.

Our recombination and transformation experiments have been successful, and we have conducted monoclonal experiments.
We conducted PCR experiments on the bacterial solution.

We obtained the sequencing results of the plasmid, demonstrating the successful construction of plasmid pGIP-LOV-VP16.
We conducted PCR experiments and based on the results, re amplified and recovered plasmids suspected to have errors during the recombination process.

We have carried out many PCR experiments, gel electrophoresis experiments and recovered DNA.
We conducted monoclonal experiments on pGIP-LOV-VP16.
We discussed the specific parameters of monoclonal experiments.

We conducted PCR experiments on two DBD fragments. The experimental results were good, and we subsequently conducted DNA recovery and homologous recombination experiments. DBD and pGIP DBD fragments were recovered and concentration measurements were conducted. We used the official website information of iResearch Biotech to calculate multiple parameters in the recombination experiment and conducted experiments.
We conducted multiple plasmid transformations and monoclonal experiments.

We obtained the sequencing results of the plasmid, demonstrating the successful construction of plasmid pGIP-GI-Gal4-DBD.
We conducted bacterial liquid PCR experiments on the monoclonal pGIP-GI-Gal4-DBD.

We conducted multiple cell plating, cell passaging, and cell transfection experiments.
In addition, we performed pGIP Luciferase plasmid sequencing.

We conducted multiple cell plating and passage, monoclonal, and cell transfection experiments.
We conducted cell recovery and plasmid extraction experiments, and measured the plasmid concentration (pcDNA-GI-Gal4, Renilla, pcDNA3.1).
We conducted a dual luciferase reporter assay to determine the activity of luciferase.
Result：GIP promoter are induced by glucose and the expression level rises along with the raise of the concentration of blood glucose.

The figure shows the fluorescence detection results at different concentrations of glucose culture medium.
The blue light instrument was sent to our laboratory.

We conducted multiple experiments on cell recovery, cell passaging and plating, and cell transfection.
We detected the status of 293T cells under a microscope.
We conducted a dual luciferase reporter assay to determine the activity of luciferase.

The figure shows shows the fluorescence detection results under different ratios of GAL4- VP16 to promoter.

We detected the status of 293T cells under a microscope.
We conducted multiple experiments on cell recovery, cell plating and passage, and cell transfection.
We conducted a dual luciferase reporter assay to determine the activity of luciferase.

We conducted research on blue light instruments and tested relevant data. After confirming the safety and accuracy of the blue light instrument and familiarizing ourselves with its operation, we began to conduct experiments using the blue light instrument.

We have organized the existing experimental results and records.
We had our final large-scale communication with the modeling team. Continue conducting cell experiments.

 This figure shows that blue light can stimulate the formation of an active transcription factor from GI-Gal4 and LOV-VP16.

We have had multiple discussions regarding the content of the wiki.
Continue conducting cell experiments.

The above figure shows our experiment exploring the optimal concentration for miRNA to exert inhibitory effects.

The above figure shows our experiment exploring the optimal concentration for sponge to exert its inhibitory effect.

We discussed the content of the wiki and identified the highlights of the experimental group.
We conducted multiple experiments on cell recovery, cell plating and passage, and cell transfection.
We conducted a dual luciferase reporter assay to determine the activity of luciferase.

The above figure shows our experiment exploring the inhibitory effect of sponge regulation by glucose concentration.

We have checked the content of the wiki and corrected any errors that occurred.