July 2021
WEEK3
7.13
- Prepare for plasmid transformation.
7.14
- Plasmid transformation of Part4(wild-type BLP-7 + linker +27*ELP) into E.coli.
- Culture the transformed bacteria on the plate with ampicillin added and liquid medium LB with ampicillin.
7.15
- Culture the transformed bacteria on the plate with ampicillin.
7.16
- Plasmid transformation of Part2(optimized-type BLP-7 + linker +27*ELP) into E.coli.
7.17
- Plasmid transformation of Part 2, 7(wild-type TLR-2 competitive inhibitor + linker + 27ELP), 8(mutational TLR-2 competitive inhibitor + linker + 27ELP) into E.coli. Pick monoclones from plate of Part 4.
WEEK4
7.18
- Pick monoclones from plates of Part 2, 4, 7, 8.
- Keep 4 tubes of monoclones(Part 2, 4, 7, 8) in shaker at 45 centigrade to induce E.colito express the target protein.
7.19
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8, and analysis the samples with SDS-PAGE.
7.20
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8, and analysis the samples with Western Blot.
7.21
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8, and analysis the samples with Western Blot.
7.22
- Prepare 4L LB medium.
7.23
- Induce E.coli to express the target protein by infrared light.
- Induce E.coli to express the target protein at 45 centigrade, and purify the protein by Ni-column.
7.24
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
WEEK5
7.25
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
- Purify the target protein by Ni-column.
7.26
- Purify the target protein E.coil by Ni-column.
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8, and perform Western Blot.
7.27
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform Western Blot.
- Induce E.coli to express the target protein at 45 centigrade.
- Prepare 3L LB medium.
7.28
- Lyse the induced(by infrared light for 40, 50, 60, 70 minutes) and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
- Analysis the target protein on SDS-PAGE.
7.29
- Induce E.coli to express the target protein at 45 centigrade for 3, 6, 9, 12 hours.
- Lyse the induced(by infrared light for 40, 50, 60, 70 minutes) and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
7.30
- Lyse the induced(by infrared light for 40, 50, 60, 70 minutes) and non-induced E.coli with Part2,4,7,8 and perform SDS-PAGE and Western Blot.
- Perform SDS-PAGE of the target protein.
7.31
- Lyse the induced(by infrared light for 40, 50, 60, 70 minutes) and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
August 2021
WEEK6
8.1
- Prepare solution needed.
8.2
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
- Purify protein with FPLC.
- Purify the target protein.
8.3
- Induce E.coli to express target protein at 45 centigrade.
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
- Perform SDS-PAGE of the purified target protein.
- Prepare for experiment of ELP assembly.
8.4
- Test the anaerobic bag.
- Purify the sample of Part2 and 4 with FPLC.
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
8.5
- Lyse the induced and non-induced E.coli with Part2, 4, 7, 8.
- Purify protein with FPLC.
8.6
- Perform SDS-PAGE with sample collected yesterday.
- Purify the sample with FPLC.
8.7
- Purify the sample of Part4 with FPLC.
- Culture the transfected bacteria on the plate with ampicillin.
- Perform BCA and SDS-PAGE to determine the concentration of E.coli induced (temperature at 45 centigrade for 24､27､30 and 33 hours) protein.
WEEK7
8.8
- Perform SDS-PAGE to determine the concentration of sample.
- Induce Part2 by infrared light for 40, 50, 60, 70 minutes and then Perform SDS-PAGE, and get a unclear band between 10KDa and 15KDa marker.
8.9
- Purify the sample of Part7 with FPLC.
- Perform SDS-PAGE and Western Blot of Part2 induced by infrared light for 40,50,60,70 minutes.
8.10
- Perform BCA test and Western Blot.
- Perform SDS-PAGE of samples of Part4 and Part7 again.
- Test the anaerobic and prepare and culture the P.acnes on the plate with heart and brain extract.
- Pick monoclones from the plates of Part2.
- Induce E.coli to express the target protein at 45 centigrade for 24, 27, 30 and 33 hours.
8.11
- Purify the sample of Part8 with FPLC.
- Pick monoclones from the plates of Part8.
- Culture the transfected bacteria on the plate with ampicillin and induce E.coli to express target protein at 45 centigrade.
- Perform Western Blot of samples of Part 2.
8.12
- Perform SDS-PAGE and Western Blot of Part 2 induced by temperature.
- Test ELP aggregation.
- Prepare solution needed.
8.13
- Purify the sample of Part2 with FPLC.
- Prepare solution needed.
- Culture the transfected bacteria on the plate with ampicillin and induce E.coli to express target protein at 45 centigrade.
- Pick monoclones from plate of Part 2 and culture at 37 centigrade.
8.14
- Perform SDS-PAGE of purified Part 2.
WEEK8
8.15
Purify the sample of Part7 with FPLC.
Perform Western Blot.
Learn about protein purification processes.
8.16
Perform SDS-PAGE of purified Part 7 yesterday.
Dynamic light scattering experiment ELP aggregation pre-experiment,size between 10 and 1000nm, Zeta potential from defeat 1.6 volts to defeat 9.8 volts,forms a visible precipitate.
Prepare LB medium.
Pick monoclones from plates.
8.17
Culture the Part4 transfected bacteria,spread plate with Part1､2､4､7 and 8 transfected bacteria, failed.
Purify the sample of Part7 with FPLC.
Inoculate the Part8 transfected bacteria.
Renaturation of ELP, failed.
Dynamic light scattering experiment ELP aggregation, no visible precipitate.
Induce Part4 expression and preserve P.acnes.
8.18
Perform SDS-PAGE of the purified Part7,unclear stripe.
Renaturation of ELP, failed.
Through A350 to test ELP aggregation, obvious effect.
Induce Part4 to express.
8.19
Purify the sample of Part2 and 8 with FPLC.
Prepare 4L LB medium.
Preserve P.acnes.
8.20
Purify the sample of Part8 with FPLC.
Prepare 4L LB medium and inoculate Part7.
Spread plate.
8.21
Induce Part2 and 7 at temperature of 45 centigrade.
Perform SDS-PAGE of Part7 and 8.
Inoculate P.acnes.
Prepare solution needed.
WEEK9
8.22
- Purify the sample of Part7 with FPLC.
8.23
Perform BCA to test the concentration of protein.
Culture P.acnes and Part4.
Purify the sample of Part2 with FPLC.
Perform SDS-PAGE of Part7 and 2.
Transfect part3, activate the ECN and cleanout the nickel column.
8.24
Transfect Part3.
Purify the sample of Part7 with FPLC.
Inoculate Part8 in the LB medium.
Prepare solution needed.
Pick monoclones from plate of Part2 and 4.
Induce Part 4 at temperature of 45 centigrade.
8.25
Purify the sample of Part4 with FPLC.
Inoculate Part4 in the LB medium and culture.
Prepare LB medium.
8.26
Purify the sample of Part8 with FPLC.
Inoculate Part8 in the LB medium and culture.
Learn about ITC, culture P.acnes.
8.27
Perform BCA and SDS-PAGE to test the concentration of Part7 and 8.
Learn about ITC.
Repair the nickel column.
8.28
Purify the sample with FPLC.
BCA test.
WEEK10
8.30
Perform BCA and SDS-PAGE to test the concentration of samples collected before.
Try new ways of culturing P.acnes.
8.31
Purify the sample of Part3 with FPLC and Perform SDS-PAGE.
Induce Part 2 at temperature of 45 centigrade, culture P.acnes.
Pick monoclones from plate of Part7 and 8.
September 2021
9.1
Transfect the mutational-27 ELP.
Sample sequencing, transmission electron microscopy.
Purification of Part2.
Culture Part4 sample.
9.2
Perform SDS-PAGE of Part2 and 3.
Test the ITC condition of PBS.
Keep Part4 at 45 centigrade to induce E.coli express target protein.
Culture Propionibacterium acnes.
Perform BCA to test the concentration of protein in lysed E.coli with Part3(8.30) and collected Part2(9.1).
9.3
Purify the sample of Part4 with FPLC.
Observe P.acnes.
Test and finalize the ITC conditions.
9.4
- Purification of Part4 sample.
WEEK11
9.5
- Induce Part2 at temperature of 45 centigrade.
9.6
- Purify the sample of Part7 with FPLC.
9.7
- Perform SDS-PAGE of Part2, 3 and 7, perform part of DMS.
9.8
- Continue practicing DMS.
9.9
Pick monoclones from the plates.
Transmission electron microscopy.
9.10
Culture Part4 sample.
Perform antibacterial test.
Perform PCR of Part7 and 8.
Perform SDS-PAGE, purification and measure the concentration.
9.11
Prepare 4L LB medium and solution needed.
Transfect the Part7.
WEEK12
9.12
Transfect Part7’ and 8’, culture them on the shaking table as well as culture by plate scribing method.
Inoculate Part 7’ in the LB medium and culture it at 37 centigrade.
Prepare 4L LB medium.
Purification of Part4 sample.
Frozen Part4 protein.
9.13
- Perform antibacterial test (use E.coli and P.acnes).
9.14
Purify the sample of Part7’ with FPLC.
Observe the results of antibacterial test.
9.15
- AFM sample diluted 10 times in the condition of heated or not heated.
9.16
- AFM sample in the condition of heated for 20 mins.
9.17
- Transfect the wild-type TLR2/Part5.
9.18
- Prepare 4L LB medium.
WEEK13
9.19
- Transfect the Part5, 6 and culture on the shaking table and incubator.
9.20
- Transfect the Part5, 6.
9.22
- Prepare 4L LB medium.
9.23
Pick monoclones from plate of Part5 and 6.
Inoculate Part6 in the LB medium and culture it at temperature of 37 centigrade.
9.24
Induce Part6 at temperature of 45 centigrade.
Purify the sample of Part5,6 with FPLC.
9.25
Inoculate Part5 in the LB medium and culture it at temperature of 37 centigrade.
Prepare 4L LB medium.
Antibacterial test.
WEEK14
9.26
- Perform BCA and SDS-PAGE to test the concentration of Part2 and Part4 protein.
9.27
- Observe the results of antibacterial test and SDS-PAGE.
9.28
Perform SDS-PAGE of samples collected before.
Inoculate Part6 in the LB medium and culture it at the temperature of 37 centigrade.
9.29
Prepare 4L LB medium.
Inoculate Part5 and 6 in the LB medium and induce at temperature of 45 centigrade.
9.30
Purify Part2 protein again.
Purify the sample of Part5,6 with FPLC.
Perform SDS-PAGE of samples of Part5, 6 induced or not.
October 2021
10.1
Perform BCA and SDS-PAGE to test the concentration of Part2 Purify(9.30).
Prepare 4L LB medium.
Pick monoclones from plate of Part5 and 6.
Induce Part 5 at temperature of 45 centigrade.
10.2
Purify the sample of Part5, 6 with FPLC.
Perform SDS-PAGE of samples of Part5, 6 induced or not.
WEEK15
10.3
Inoculate monoclones Part5 and 6.
Prepare solutions needed.
10.4
- Pick monoclones from plate of Part5, 6 and culture at temperature of 37 centigrade.
10.6
Inoculate Part5, 6 and culture at temperature of 37 centigrade, Perform SDS-PAGE of samples of Part5,6 induced or not.
Prepare solutions needed.
10.7
Perform SDS-PAGE to test the concentration of Part2 purified (9.30) again.
Induce Part5 and 6 at temperature of 45 centigrade, perform SDS-PAGE of samples induced at different temperature, Part5 at 37 centigrade, Part5 at 45 centigrade, Part6 at 37 centigrade and Part6 at 45 centigrade.
Prepare solutions needed.
10.8
Perform SDS-PAGE of samples collected before.
Inoculate monoclones Part5, 6 and culture at temperature of 37 centigrade.
10.9
Induce Part5 and 6 at temperature of 45 centigrade.
Perform SDS-PAGE of samples collected before.
Inoculate monoclones Part5, 6.
WEEK16
10.10
Inoculate monoclones Part5, 6.
Prepare the samples induced at different temperature, part 5 at 37 centigrade, Part5 at 45 centigrade, Part6 at 37 centigrade and Part6 at 45 centigrade, ECN for SDS-PAGE.
Transfect ECN.
Check the results of SDS-PAGE in 10.9.
Prepare solutions needed.
10.11
Check the results of SDS-PAGE in 10.10.
Part5, 6 samples sequencing.
10.12
Culture the Part5 transfected bacteria.
TransfectPart5, 6.
Inoculate monoclines Part5, 6.
The sequencing in 10.11 has no signal.
10.13
Induce Part5 at temperature of 45 centigrade.
Transfect Part5, 6.
10.14
Purify the sample of Part5 with FPLC.
Induce Part5 and 6 at temperature of 45 centigrade.
10.15
- Perform SDS-PAGE of samples collected in 10.14.
10.16
Prepare the samples induced at different temperature, Part5 at 37 centigrade, Part5 at 45 centigrade, Part6 at 37 centigrade and Part6 at 45 centigrade and perform SDS-PAGE.
Check the results of SDS-PAGE in 10.15.
WEEK17
10.17
- Check the results of SDS-PAGE in 10.16.