In our first week in the laboratory, we reviewed and learned the required experimental procedures and began growth testing of various chassis strains including yeast laboratory strains and engineered strains. We also conducted a comprehensive inventory of the laboratory.
We selected engineered strains CEN.PK113-7d and BY4741 to further explore their synthesis effects. We performed chemical transformation with 6 high-efficiency expression plasmids: no sp, SUC2 sp, INU1 sp, PHO5 sp, SCW11 sp, and HECH sp for induced expression, and used WB to detect whether LL-37 was secreted. Meanwhile, we began preparing for LL-37 single-copy site-specific integration. We also conducted literature research on different functional protease genes in Saccharomyces cerevisiae.
This week, we constructed the single-copy integration plasmid Topo-Homo Ty1-URA3-LL37 using PCR, transformed it into E. coli and verified it (colony PCR). We induced CEN.PK113-7d and BY4741 strains, sampling at 12-hour intervals to explore induction time. During this period, we reviewed literature on LL-37's antimicrobial mechanism and methods.
With preliminary experiments successfully completed, we selected CEN.PK113-7d as our main chassis cell. Starting this week, we entered multi-faceted genetic engineering experiments. First, we found 17 corresponding protease gene deletion strains in the laboratory's YKO strain library (gal80△, pep4△, cym1△, yps1△, ysp3△, prb1△, prc1△, cps1△, bar1△, hrd1△, dap2△, kex2△, ire1△, ubp3△, der1△, ssa1△, ssb1△) (from mid-March literature research), performed induced fermentation and qualitatively observed LL-37 production using WB. Second, we constructed and verified reporter plasmids for subsequent promoter modification. We also constructed the multi-copy integration plasmid Ty1-LoxP-URA3-pGAL1-LL37.
This week we used error-prone PCR to amplify and mutate GAL1 and GAL10 promoters, collected the GAL promoter mutation library, expanded and extracted plasmids for sequencing. We also transformed the multi-copy integration plasmid into yeast for expression and completed WB detection. We designed primers for knocking out PEP4 and YPS1.
We re-mutated the GAL promoter. We attempted to knock out PEP4 and YPS1 separately in CEN.PK113-7d strain and verified successful knockout using colony PCR. We also prepared mobile phases required for HPLC, disrupted samples from the 17 protease gene deletion strains after fermentation, and performed sample loading at week's end. We also designed primers for knocking out multiple genes including CYM1, YPS3, and BAR1.
This week we conducted high-throughput testing of promoter mutants, inoculated fluorescence kinetic curves, and analyzed data over the weekend. We also performed marker recycling for URA3 in CEN.PK113-7d ura3△.
Beginning May, we organized preliminary data and retested secretion in the 17 protease gene deletion strains. We began CYM1 and BAR1 gene knockout and verified marker recycling in pep4△ and yps1△ by streaking plates.
This week, we first performed inhibition zone experiments (E. coli) with chemically synthesized LL-37 samples, recovered plasmids from pep4△ and yps1△, began knocking out BAR1 and HRD1 and completed gene knockout verification before week's end, then transformed the pre-extracted pY26-kanMX-Cre plasmid for marker recycling.
We also began reviving the YKO strain mixed pool for subsequent irrational screening of the YKO strain library.
We verified marker recycling in pep4△ and cym1△. Since the previous marker recycling for yps1△ and prb1△ was unsuccessful, we re-transformed Cre plasmid this week and completed strain preservation for bar1△ and hrd1△. Meanwhile, we performed high-throughput fermentation of the YKO mixed pool, designed HAF1 overexpression primers and gal80△ knockout primers and performed knockout mid-week; designed the pY26-GAL1-LL37-eGFP plasmid for subcellular localization.
We also conducted literature research, finding vacuolar protease gene deletion strains in the YKO strain library (ape1△, ape3△, yps7△, ysp3△, vps10△, vps1△, vps15△, vps8△, vps35△).
This week, we first verified GAL80 gene knockout and prepared CEN.PK113-7d ura3△.gal80△ competent cells for marker recycling; extracted the his-tagged pESC-GAL1-LL37 production plasmid.
We used 96-well plates for liquid antimicrobial testing to determine the Minimum Inhibitory Concentration (MIC) of LL-37 for dry lab model prediction.
This week we mainly conducted construction and verification of various plasmids. We constructed localization plasmids with α-factor signal peptide pESC-GAL1-α-factor-LL37 and pESC-GAL10-PLN1-mCherry, verified plasmids Topo-Ty1-KanMX-LL37, pESC-GAL1-LL37-GAL10-LL37, pESC-GAL1-LL37-eGFP and extracted for storage. We also completed transformation of two plasmids pY26-URA3-Cre and pESC-GAL1-LL37(6his) in CEN.PK113-7d ura3△.gal80△. We designed new gene knockout primers (vps10△, vps8△, ape1△, ysp3△).
This week we continued plasmid construction, checked last week's transformation results, reconstructed plasmid pESC-GAL1-α-factor-LL37, and constructed, verified, and extracted multiple plasmids.
We also collected growth kinetics data for CEN.PK113-7d ura3△.gal80△::loxp-KanMx, pESC-GAL1-LL37 and prepared different carbon sources for subsequent fermentation condition optimization.
Starting this week, our experimental team accelerated the experimental process, dividing into scientific exploration and production fermentation groups. The exploration group conducted strain construction and genetic engineering experiments; the fermentation group optimized purification methods and other downstream technologies. The exploration group verified marker recycling in 4 sorting protein strains (pep1△, ysp3△, ape1△, vps8△) and performed plasmid elimination, also induced co-localization plasmids with α-factor, taking samples at intervals (12h, 24h, 36h, 48h) for fluorescence microscopy observation. The fermentation group explored sample processing methods, performed WB detection of fermentation with different carbon sources, and measured residual sugar and gene expression coupling during CEN.PK113-7d ura3△.gal80△ fermentation.
This week we continued the multi-copy integration started earlier, performed marker recycling for CEN.PK113-7d ura3△.ty1△::loxP-kanMX-GAL1-LL37, and re-knocked out GAL80. We also revived and activated strains from 96-well plates under different conditions, then diluted and measured OD600. We simultaneously began irrational screening of the YKO strain library, activating A1-D12.
Considering the final application of LL-37, we began downstream purification processing, attempting purification using Ni columns with gradient elution. Meanwhile, we performed high-salt low-pH medium gradient testing (0, 0.15, 0.3, 0.45, 0.6, 0.75, 0.9 M NaCl), monitoring growth and measuring supernatant. We also conducted shake flask fermentation with high-salt low-pH conditions and timed sampling (36h, 72h, 108h, 120h).
Strain construction continued with verification of last week's GAL80 knockout, beginning marker recycling and plasmid elimination, and starting MIG1 knockout.
Last week's Ni column purification failed, possibly due to low column efficiency. This week we tried elution with a new Ni column. We inoculated YKO irrational screening strains in 24-well plates for fermentation for subsequent HPLC detection. We also performed WB detection on disrupted fermentation samples from last week, testing both supernatant and whole cell lysates.
Based on earlier localization plasmids, we further constructed four co-localization plasmids (cell membrane, vacuole, ER lumen, Golgi apparatus) and PCR verified multiple gene knockouts (mig1△, pep4△, prb1△, ysp3△, yps1△).
We PCR verified co-localization plasmids and reconstructed the ER co-localization plasmid pESC-LL37-eGFP-GAL10-KAR2-mCherry, attempted to construct the his-tag-free dual expression cassette plasmid pESC-GAL1-LL37-GAL10-LL37, and ligated plasmid Topo-Ty1-KanMX-GAL1-LL37-GAL10-LL37. Unfortunately, four previously successfully knocked-out strains became contaminated (pep4△, prb1△, yps1△, ysp3△), so we purified and preserved them.
We also optimized extraction methods, beginning multiple rounds of LL-37 extraction with strong acid high-salt solutions, later trying multiple rounds of elution with urea. We performed fermentation of YKO library E1-H12 and ultrafiltration concentration.
Last week's co-localization plasmid construction failed, so we tried synthesizing co-localization fragments using fusion PCR and gel extraction. We also performed MIG1 knockout and verification, and organized single knockout strains.
Even with the new Ni column, LL-37 still couldn't bind, so this week we tried cascade purification of His-tagged LL-37. We also tried different surfactants for extraction (SDS, Tween-20, TritonX 100, LDS, CHAPs) and high-pressure homogenization extraction.
We also compared antimicrobial effects of LL-37 and NisinA, and prepared to test antimicrobial activity of dry lab predicted variants.
This week's experimental focus was using the D2P system to synthesize four dry lab predicted variants plus original LL-37 and 6*His-LL37. We also began constructing double knockout strains.
For purification, previous attempts had limited effect. This week we tried magnetic bead purification with Coomassie brilliant blue staining. We also performed WB testing of surfactant extraction effects, followed by dialysis removal and ultrafiltration to preserve samples.
This week's experiments proceeded smoothly. After PCR verification of double knockout strains, we verified plasmid elimination by streaking and preserved strains. We also began constructing triple knockout strains. We performed WB detection of last week's nickel magnetic bead purification effect, used nickel magnetic beads to purify D2P products, and performed ultrafiltration desalting after dilution.
This week we transformed successfully constructed double knockout strains with LL-37 production plasmids and began constructing SUMO-tagged pESC-GAL1-MFα-SUMO-LL37 plasmid and multi-copy integration plasmid Topo-TY1-loxP-KanMX-GAL1-LL37-GAL10-LL37. We activated and fermented SUMO-transformed yeast. We also prepared promoter sequences and antimicrobial peptide sequences.
This week we again attempted to construct co-localization plasmid backbone and performed marker recycling for triple knockout strains. We used WB to verify SUMO tag secretion and timed D2P samples, and conducted D2P antimicrobial experiments.
These two weeks experiments stagnated. SUMO tag secretion verification failed, with no product detected even in cell pellets. We suspected problems during plasmid transformation, so this week we repeated fermentation testing following original procedures. Triple knockout strain marker recycling verification by streaking was also unsuccessful, so we continued Cre plasmid transformation this week. Construction of four co-localization plasmids continued.
However, we had new experimental directions this week. We changed our promoter modification strategy, directly PCR amplifying strong promoters TEF1 and TDH3 from synthesized sequences. We also performed plate killing tests on D2P's four variants and two original samples.
We began preparing materials for writing.
This week we continued constructing triple knockout strains. Currently two triple knockout strains are in plasmid elimination stage, while the other two are being reconstructed. We performed medium composition testing and collected growth kinetics data for analysis with dry lab. We also performed WB detection of last week's SUMO fermentation and colony counting for killing experiment plates.
This week we began constructing quadruple knockout strains and performed multi-copy integration. We checked production plasmid transformation of previously constructed double and triple knockout strains, and tested modified promoters with fluorescent proteins. We reconstructed the SUMO plasmid, changed SUMO position and sequenced it, and began a new round of fermentation.
This week, we mainly prepared double, triple, and quadruple knockout strains for fermenter runs, while organizing all experimental data and preparing and revising materials needed for the wiki.