| Date | System | Activities | Results |
| July 1 | System 1 | Prepare plasmid with CrCPR, LjIFS, GmHID, perform codon optimization, and synthesize genes | Plasmids ready for transformation into E. coli BL21 |
| July 2-4 | System 1 | Transform plasmid into E. coli BL21 and screen for positive clones on Amp LB plates | Positive clones with plasmids obtained |
| July 5 | System 1 | sequencing to verify gene insertion | Successful verification of gene insertion |
| July 6-8 | System 1 | Begin fermentation of BL21 strain in LB media | Fermentation process established |
| July 9 | System 1 | Measure absorbance values of genistein standards to create the standard curve | Standard curve established for calculating genistein concentration |
| July 10 | System 1 | Measure absorbance values from the production strain and calculate genistein concentrations | Initial genistein production data obtained |
| July 11-12 | System 1 | Truncate the first 21 amino acids of LjIFS and compare genistein production before and after truncation | Results show the impact of truncation on genistein production |
| July 13-14 | System 1 | Modify truncated LjIFS with N-terminal tags (17A, KKK, HHHH) and compare their effects on genistein yield | Comparison of different tag effects on IFS activity |
| July 15 | System 1 | Test the addition of OmpAL to the N-terminus of CrCPR and measure its effect on genistein synthesis | Impact of OmpAL on genistein production evaluated |
| July 16 | System 2 | Construct plasmids containing acsAB and acsCD, transform into E. coli BL21 and screen for positive clones | BL21/AcsAB and BL21/AcsAB-AcsCD strains successfully constructed |
| July 17-18 | System 2 | Measure intracellular and extracellular cellulose production, compare the synthesis capabilities of both strains | Analysis of cellulose production differences between the strains |
| July 19-21 | System 2 | Process the produced cellulose into facial masks, test water retention and tensile strength | Data on water retention and tensile strength of cellulose masks collected |
| July 22-23 | System 2 | Analyze data from water retention and tensile strength tests | Results show high water retention and strong tensile properties of cellulose masks |
| July 24 | System 3 | Construct blue light-inducible reporter system pSB-pDawn-mRFP, transform into E. coli DH5α | Positive clones for reporter system obtained |
| July 25 | System 3 | Transform pSB-pDawn-mRFP，suicide system into E. coli BL21 | BL21 system verification successful |
| July 26 | System 3 | Test the fluorescence expression of the blue light-inducible reporter strain | Fluorescence data obtained to confirm blue light induction |
| July 27-29 | System 3 | Construct blue light-inducible suicide system pSB-pDawn-mazF, transform into E. coli DH5α and test | DH5α system verification successful |
| July 30-31 | System 3 | Transform pSB-pDawn-mazF suicide system into E. coli BL21 and perform blue light exposure tests | BL21 suicide system test results collected |
| August 1-2 | System 1 | Analyze the effects of LjIFS truncation, N-terminal modifications, and OmpAL addition on genistein production | Optimized conditions for maximizing genistein yield determined |
| August 3-5 | System 1 | Summarize and organize experimental data on genistein production | Final genistein production data compiled for report |
| August 6-7 | System 2 | Conduct final cellulose production experiments, collect and analyze cellulose yield data | Final cellulose production data collected and evaluated |
| August 8-10 | System 3 | Summarize the results of blue light-induced suicide system tests and prepare visual data | Report finalized with summarized results and graphical representations |
| August 11-12 | All Systems | Process data, create visual figures, and draft final report | Complete data analysis and final report ready for submission |
Objective: Construct the genistein-producing strain BL21/IFS-CPR-HID by integrating plant-derived genes (CrCPR, LjIFS, GmHID) into E. coli, and enhance genistein production by truncating and modifying the N-terminal of LjIFS and CrCPR.
Procedure:
Construct a plasmid containing CrCPR, LjIFS, and GmHID, and transform it into E. coli BL21 to create the genistein-producing strain.
Use a genistein standard curve to measure absorbance values and calculate genistein concentrations from production strains.
To increase genistein yield, truncate the first 21 amino acids of LjIFS and compare the production before and after truncation.
Further modify the truncated LjIFS by adding N-terminal tags (17A, KKK, HHHH) and assess their impact on genistein yield while keeping CrCPR and GmHID constant.
Add the OmpAL tag to the N-terminus of CrCPR and evaluate whether it enhances genistein synthesis efficiency by improving CrCPR secretion.
Results: Investigate the impact of different N-terminal tags and truncation on genistein production, optimizing the expression and functionality of LjIFS and CrCPR.
System 1:Laboratory notebook（Total 43 pages）
Objective: Construct cellulose-producing strains BL21/AcsAB and BL21/AcsAB-AcsCD, and compare their cellulose synthesis capabilities.
Procedure:
Construct plasmids containing acsAB and acsCD cellulose synthase genes, and transform them into E. coli BL21 to form cellulose-producing strains.
Measure the cellulose production of the strains by comparing intracellular and extracellular cellulose yields.
Perform small-scale experiments to process the produced cellulose into facial masks and test their water retention and tensile strength.
Results: Compare the cellulose production between different strains, evaluate the performance of cellulose-based facial masks, and analyze intracellular and extracellular cellulose production.
System 2：Laboratory notebook（Total 24 pages）
Objective: Construct and test blue light-inducible reporter and suicide systems.
Procedure:
Construct the blue light-inducible reporter strain pSB-pDawn-mRFP, first transform it into E. coli DH5α for verification, and then transform it into E. coli BL21 for final functional testing , and test its fluorescence expression to verify the effectiveness of blue light induction.
After constructing the pSB-pDawn-mazF blue light-inducible suicide system, first transform it into E. coli DH5α for verification, and then transform it into E. coli BL21 for final functional testing under blue light exposure to monitor cell death.
Results: The blue light-inducible suicide system effectively induces cell death under blue light exposure in BL21, significantly reducing cell survival compared to control groups.
System 3：Laboratory notebook（Total 34 pages）
