Week 1 - (Feb. 27th-Mar.5th)
Learned and mastered the basic experimental methods. Expanded pcDNA3.1(+) plasmids in DH-5α Escherichia Coli, extracted the plasmids and measured the concentration.
Week 2 - (Mar.6th-Mar. 12th)
The method of obtaining full-length GnRH and truncated GnRH (GNRH-C) plasmids was discussed and determined. We provided full-length GnRH fragments to the company and they were synthesized by the company. A primer was designed to clone the GnRH-C fragment (GnRH-GAP) from the full-length Signal peptide-GnRH-GAP, and the fragment was connected to pcDNA3.1(+) vector by enzyme linking. Then the PCR primers were designed and ordered.
Week 3 - (Mar. 13th-Mar.19th)
After the primers arrived, we started to construct the pcDNA3.1(+)-GnRH-C plasmids. Firstly, we obtained GnRH-C fragments from the full-length GnRH plasmids by PCR. Then we selected the Nhel and Xhol sites on the pcDNA3.1(+) vectors for restriction enzyme digestion and link with GnRH-C fragments. Agarose gel electrophoresis showed that the results of plasmids construction did not get the expected bands. The experimental results were not ideal.
Week 4~5 - (Mar.20th-Apr.2nd)
The construction of pcDNA3.1(+)-GnRH-C plasmid failed and the ideal result was not obtained, which was suspected to be due to the experimental conditions, experimental procedures and operational problems. Therefore, in these two weeks, we explored better experimental conditions, optimized experimental operations, and continued to construct pcDNA3.1(+)-GnRH-C plasmid by eliminating experimental errors caused by operations as much as possible. However, the plasmid was not successfully constructed after improving the experimental conditions.
At the same time, we prepared for subsequent experiments, checked whether laboratory consumables were complete, and prevented the progress of the experiment from being difficult to advance due to external reasons.
Week 6 - (Apr.3rd-Apr.9th)
The complete pcDNA3.1(+) vector was used and amplified.
At the same time, the method of constructing pcDNA3.1(+)-GnRHR plasmid was designed. After discussion, PCR was adopted to amplify GnRHR fragment from human genome by designing suitable primers, and the fragment was connected to pcDNA3.1(+) vector by enzyme cutting and linking. The design and order of primers were finished.
Week 7 - (Apr.10th-Apr.16th)
Primers for the construction of GnRHR arrived, and pcDNA3.1(+)-GnRHR and pcDNA3.1(+)-GnRH-C plasmids were constructed this week. The relevant fragments were obtained from the template by PCR, and then linked to the pcDNA3.1(+) vector skeleton by enzyme digestion. The results of plasmid construction were tested by agarose gel electrophoresis, but the expected results were not obtained, so the plasmid construction failed.
Week 8 - (Apr.17th-Apr.23rd)
After discussion and consultation with teachers, it was speculated that the failure might be caused by the low success rate of the selected cleavage site. In order to promote the progress of the experiment, we chose the method of homologous recombination with a higher success rate to connect the vector and fragment, and redesigned and ordered the primers.
After the primers arrived, PCR was conducted and the vector and fragment were connected. The results of plasmid construction were detected by agarose gel electrophoresis. The pcDNA3.1(+)-GnRH-C plasmid had expected bands, and the sequencing results were also correct, indicating successful plasmid construction.
However, pcDNA3.1(+)-GnRHR still did not get the expected bands, and this plasmid construction failed.
Week 9 - (Apr.24th-Apr. 30th)
We started constructing pcDNA3.1(+)-GAL4-VP64-NLS plasmid and pGL4.35-3×HA-9×GAL4UAS-KRAB-Degron-NLS plasmid, constructed plasmid map, designed and ordered plasmid used for recombination.Obtained relevant fragments by PCR from the plasmid containing the desired elements, and connected them to the vector by homologous recombination.
After the arrival of the primers, the construction of pcDNA3.1(+)-GAL4-VP64-NLS plasmid was completed, the expected band was detected by agarose gel electrophoresis and sent to the company for sequencing, the results were correct and the plasmid construction was successful.
The repeated construction of the pcDNA3.1(+)-GnRHR plasmid failed.In order to advance the experiment, the plasmid was purchased from the company.
Week 10 - (May. 1st-May.7th)
The pGL4.35-9×UAS-GAL4-KRAB-NLS plasmid was successfully constructed. At the same time, we successfully amplified the pcDNA3.1(+)-GAL4-VP64-NLS and pGL4.35-3×HA-9×GAL4UAS-KRAB-Degron-NLS plasmid.
Week 11 - (May. 8th-May.14th)
Determine methods to detect whether the pcDNA3.1(+)-GnRH plasmid can transcribe and translate the GnRH protein in cells. The GnRH detection system was constructed by transient transfection of pcDNA3.1(+)-GnRHR plasmid in CHO-AEQ cells, and the change of calcium flow was detected by Sirius single-tube chemiluminescence detector. The conclusion was obtained, and the work of the system was verified by GnRH protein. After successful verification, the supernatant of 293T cells transfected with pcDNA3.1(+)-GnRH plasmid was used to detect changes in intracellular calcium flow, and to detect whether pcDNA3.1(+)-GnRH plasmid could trantranate and translate GnRH protein in cells.
Week 12 - (May. 15th-May. 21st)
Resusitated cryopreserved CHO-AEQ cells, were found to have mycoplasma contamination during culture. The use of pesticides and resuscitation of other branched cells were used to solve the contamination problem, and the experiment was suspended to find a solution.
Week 13 - (May. 22nd-May.28th)
The plasmids needed for the experiment were amplified and extracted. To measure the cell rhythm, the HEK 293T cells were co-transfected with pcDNA3.1(+)-GAL4-VP64-NLS plasmids, pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmids and Luciferase plasmids at the concentration ratio of 1:1:1. The cell rhythm was detected by Lunicycle instrument, and the period was one day.
It was determined that the AEQ plasmids was transiently transfected into HEK 293T cells to replace the CHO-AEQ cells stably transfected with AEQ plasmids. Then we determined to detect whether the pcDNA3.1(+)-GnRH-C plasmids could transcribe and translate GnRH proteins in 293T cells. We also planed to buy GnRH standard.
Week 14 - (May. 29th-Jun. 4th)
We detected the activity of GnRHR. We cotransfected HEK 293T cells with pcDNA3.1(+)-GnRHR plasmids and AEQ plasmids, and then we detected Ca2+ current changes of these HEK 293T cells by Sirius single-tube chemiluminescence detector when different concentrations of GnRH standards were added. The ideal experimental results were not obtained. We supposed the reason was that HEK 293T cells gathered into clusters, which led to insufficient cell lysis.
Week 15 - (Jun. 5th-Jun. 11th)
Considering that Hela cells can adhere to the wall better, HEK 293T cells detected for cell rhythm and GnRHR activity were replaced with Hela cells. Then we repeated previous transfection and detection experiments. The ideal results were not found in both groups.
Week 16 - (Jun. 12th-Jun. 18th)
In the experiment of detecting the activity of GnRHR, We soaked the culture dishs in PDL to make cells better adhere to the walls of culture dishs. And we also replaced the Hela cells detected for cell rhythm and GnRHR activity with HEK 293T cells originally used.
After soaking culture dishs with PDL, we co-transfected HEK 293T cells with pcDNA3.1(+)-GnRHR plasmids and AEQ plasmids, and then we detected Ca2+ current changes of these HEK 293T cells by Sirius single-tube chemiluminescence detector when different concentrations of GnRH standards were added. The ideal experimental results were not obtained.
Week 17 - (Jun. 19th-Jun. 25th)
We detected the Cell rhythm . pCDNA3.1(+)-GAL4-VP64-NLS plasmid and pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid concentration ratios are 1:2, 1:3, 1:10, 1:30 and 1:90,and the quantitative Luciferase plasmid (the total number of plasmids remained unchanged) were transfected into 293T cells to detect the changes in cell rhythm.
Week 18 - (Jun. 26th-Jul. 2nd)
Because of the review and preparation for the final exam, the experiment was suspended and the main task was data analysis and summary.
Week 19 ~ 20 - (Jul. 3rd-Jul. 16th)
Conducted the final examination, participated in the field practice organized by the college, and did not conduct the experiment.
Week 21 - (Jul. 17th-Jul. 23rd)
In the detection of changes in intracellular calcium flow experiments, we chose to transiently transfect the AEQ and GnRH-R plasmid into HEK 293T cells with a mass ratio of 1:1, repeat the transfection and test the Ca2+ experiments. However, no ideal experimental results appeared.
To test the cell rhythm assay, we transfected pCDNA3.1(+)-GAL4-VP64-NLS plasmid, pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid into HEK 293T cells at a 1:1 mass ratio. Then Luciferase was observed by detecting the bioluminescent intensity, and Luciferase showed rhythmic oscillation.
Week 22 - (Jul. 24th-Jul. 30th)
In the detection of changes in intracellular calcium flow experiments, HEK 293T cells were replaced with Hela cells, and transfection was repeated as well as the assay. No ideal experimental results occurred.
In cell rhythm experiments, HEK 293T cells were co-transfected with pcDNA3.1 (+) -GAL4-VP64-NLS plasmid and pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid, concentration ratio of 2:1,4:1,8:1,16,16:1, and quantified Luciferase plasmid to detect the changes in cell rhythm.
Week 23 - (Jul. 31st-Aug. 6th)
In the detection of changes in intracellular calcium flow experiment, we replaced all the tested HEK 293T cells with U2OS (human osteosarcoma cells) cells, and repeated the transfection and the detection experiment. However, no ideal experimental results appeared.
Week 24 - (Aug. 7th-Aug. 13th)
For the detection of changes in intracellular calcium flow experiments, we replaced all U2OS cells with CHO cells, repeated the transfection and the detection experiments. In order to exclude the existence of experimental operational problems, we transiently transfected plasmid containing PROK 2 receptor and AEQ plasmid into CHO cells, and used PROK 2 protein dilution to detect the change in calcium fluidity, and obtained the expected results.
At the same time, we respectively transfected to two groups of HEK 293T cells plasmids containing GnRH sequence and truncated GnRH Associated Peptide GnRH sequence plasmid (GnRH-C) and culture enough time.Take the supernatant of two cell culture media instead of the original GnRH protein diluent for calcium ion mobility testing, in order to obtain the secretion of the two proteins in HEK 293T cells.
Meanwhile, we constructed the GAL4-KRAB-Degron plasmid and the GAL4-VP64-Degron plasmid by homologous recombination. Meanwhile, we constructed the GAL4-KRAB-Degron plasmid and the GAL4-VP64-Degron plasmid by homologous recombination. To explore the effect of shortening the protein half-life on the cell cycle, We designed four sets of plasmids to transfect cell ：①pcDNA3.1-GAL4-VP64-NLS plasmid and pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid; ②pcDNA3.1-GAL4-VP64-NLS Plasmid and the KRAB-Degron plasmid; ③pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid and the GAL4-VP64-Degron plasmid;and④GAL4-KRAB-Degron plasmid and the GAL4-VP64-Degron plasmid. All transfection mass ratio was 1:1 and experiments were performed.
Week 25 - (Aug. 14th-Aug. 20th)
In the detection of changes in intracellular calcium flow experiment, we again transfected the AEQ plasmid and the GnRHR plasmid into CHO cells . Ca 2 + assays were performed using the GnRH protein dilution after sufficient incubation time. The image did not show the expected kurtosis.
During this period, we modified the experimental conditions in the course of repeating the test to extend the equilibration time of the cell suspension to 1h30min and achieve the expected test results, while obtaining reproducible experimental results.
At the same time, we designed the half-life test of Degron protein by consulting relevant literature.
Week 26 - (Aug. 21st-Aug. 27th)
The cell rhythm detection experiments, detection of changes in intracellular calcium flow experiments and Degron half-life detection experiments were performed.
The experimental group jointly participated in the writing and improvement of the relevant content in the wiki team.
Week 27 - (Aug. 28st-Sep. 3rd)
Performed the Degron half-life detection experiments.
Week 28~31- (Sep. 4th-Oct. 1st)
Organize the experimental results, edit the content of various parts of the wiki, modify and improve it, and upload it to the website page.
