Part:BBa_K5261017
Bioadhesion expression pathway
In Saccharomyces cerevisiae BY4741, the promoter region of the FLO11 gene is repressed by signal regulation caused by a nonsense mutation in the transcriptional regulator Flo8p, silencing the FLO11 gene, resulting in the loss of the surface adhesion phenotype. The promoter of the FLO11 gene was replaced with the TET1 promoter linked to the FLO11 gene, enabling its efficient expression in yeast.
Overview
Here, we used the constitutive strong promoter pTEF1 to overexpress the Saccharomyces cerevisiae surface adhesion gene FLO11 to construct yeast biofilms in a hydrophobic carrier filler of MBBR-MABR membrane bioreactor. We used the CRISPR-Cas9 system to insert the pTEF1 promoter upstream of the coding region of the endogenous gene FLO11 in Saccharomyces cerevisiae BY4741. Subsequently, we performed the surface adhesion ability characterization of the Saccharomyces cerevisiae BY4741 FLO11+ strain.
Integration of the pTEF1 promoter using the CRISPR-Cas9 system
Construction of the CRISPR-Cas9 system’s integration tool
Using the CRISPR-Cas9 system requires separate construction of plasmids containing gRNA, Cas9, and donor fragments for gene integration according to the principle of homologous recombination.
First, the appropriate gRNA sequence was designed according to the gene sequence of the recombination site within -200 bp upstream of the coding region of the FLO11 gene through the CRISPR assisted-design website (https://benchling.com). The p426-SpSgH-URA3 plasmid was inserted with the BsaI enzyme to generate separate expression plasmids for gRNA1-4. Then, according to the principle of homologous recombination, homologous primers with 40 bp homologous arms were designed. The gene integration fragment pTEF1 donor was obtained by PCR from pUMRI-HO-pTEF1 plasmid, and the PCR product was concentrated by ethanol precipitation to obtain a high concentration integrated fragments.
Figure 1. The CRISPR-Cas9 integration tool. A. The p426-SpSgH-URA3 plasmid. B. The p416-Cas9-G418 plasmid. C. gRNA1-4 is designed for gene integration just upstream of the FLO11 gene. D. Integration of the pTEF1 promoter upstream of the FLO11 gene.
Electrophoresis result conveyed that the pTEF1 donor fragment (theoretical band 493 bp) was successfully obtained, and the result of sequencing showed that the gRNA expression plasmid p426-SpSgH-URA3-gRNA was successfully conducted
Figure 2. A. Electrophoresis result showing that the pTEF1 donor was successfully obtained. B. Sequencing result showing a successful construction of p426-SpSgH-URA3-gRNA plasmid.
Transforming Saccharomyces cerevisiae by CRISPR-Cas9 system integration tool
The two plasmids mentioned above and the donor were introduced into Saccharomyces cerevisiae BY4741 using lithium acetate transformation to achieve gene integration. Single clones were obtained after 3 days of culture on SC-URA+G418 agar plates. Sixteen single yeast colonies were selected as templates for colony PCR and verified by agarose gel electrophoresis.
Electrophoresis result showed two positive clones (The region flanking the recombination site upstream of the FLO11 coding region. A successfully integrated band theoretically has 729 bp, or 523 bp if weren’t integrated.)
Figure 3. Colony PCR verified the electrophoresis results of CRISPR-Cas9 gene integration. Positive results for numbers 2 and 13 (target band 729 bp)
After sequencing validation, the stably inherited Saccharomyces cerevisiae BY4741 FLO11 + strain was obtained.
Figure 4. Sequencing showed that the pTEF1 promoter was successfully integrated upstream of the endogenous FLO11 gene in Saccharomyces cerevisiae BY4741.
Characterization of the surface adhesion phenotype of the BY4741 FLO11 + strain
Enhanced cell-cell adhesion
After growing Saccharomyces cerevisiae BY4741 and BY4741 FLO11+ strains in YPD liquid medium overnight, photographs were taken and natural sedimentation of the two strains was recorded every 30 min by simultaneously standing without shaking.
Within 60 minutes, the natural sedimentation rate of Saccharomyces cerevisiae BY4741 FLO11 + strain was significantly faster than that of BY4741. Although Flo11p is thought to be associated with cell-surface adhesion, we propose that overexpression of the FLO11 gene also conferred a mild cell-cell adhesion phenotype of Saccharomyces cerevisiae BY4741 FLO11+. Therefore, we also simultaneously observed the two strains under a light microscope.
At 40× magnification， more cell-cell adhesion of BY4741 FLO11+ strains in the field of view, while wild-type BY4741 cells were dispersed; At 100× magnification, BY4741 FLO11 + cells formed large clumps of 5-30 cells
Figure 5. A. BY4741 FLO11+ strains naturally settle faster than wild-type BY4741. B. The cell-cell adhesion phenomenon was observed in the BY4741 FLO11 + strain.
Adherence to agar and plastic plates
We subsequently characterized the cell-surface adhesion properties, which included adhesion to plastic and agar.
1. Characterization of plastic surface adhesion
The cultures of the two strains were overlaid on plastic dishes made of polystyrene. After one day, pour the surface culture medium and rinse with water. The BY4741 FLO11+ strain was seen to form a radiopaque biofilm at the bottom of the plastic plate. The biofilm was stained with the ammonium oxalate-crystal violet dye solution and was observed with a light microscope
Figure 6. A. BY4741 FLO11 + strain formed a biofilm at the bottom of the polystyrene plate. B. Biofilm morphology under the microscope.
At 40× magnification, the BY4741 FLO11+ strain in the visual field formed dense biofilm, while the wild-type BY4741 cells showed a loose dispersed distribution, which suggested the ability of strain BY4741 FLO11+ to adhere to the plastic surface and form biofilm.
2. Characterization of agar surface adhesion
After the simultaneous culture of Saccharomyces cerevisiae BY4741 and BY4741 FLO11 + strains on YPD solid agar plates for 2 days, the two strains grew equally well on the agar plates (before washing).
When the plate was gently washed with the running water, the cells of the wild-type BY4741 were almost completely washed out, and the majority of the cells overexpressing FLO11 remained firmly adhered to the agar surface, which could indicate the ability of the BY4741 FLO11 + strain to adhere firmly to the agar surface.
Figure 7. BY4741 FLO11+ strain exhibited a stronger adhesion ability on YPD agar plates.
Quantitative characterization of adhesion in polypropylene 96-well plates
The YPD cultures of the above two strains were seeded in an inoculum of 100 μL OD600 =1 in wells of polypropylene 96-well plates, incubated for 60,120,180, and 240 minutes to allow sufficient adherence, then continued biofilm staining for 30 minutes by adding 50μ L of ammonium oxalate-crystal violet dye solution. After the staining, the cells were washed four times with PBS buffer to remove the excess dye solution, and finally 200μL of absolute ethanol was added to dissolve the dye solution on the biofilm in each well. The absorbance at 590 nm for each well was measured using a microplate reader. Three parallel experiments were done for each data point.
On polypropylene 96-well plates, the BY4741 FLO11+ strain tended to be significantly more adherent than the BY4741 strain
Figure 8. A. After the adherent biofilms in polypropylene 96-well plates were stained with ammonium oxalate-crystal violet dye solution, the absorbance at 590nm was quantified in a microplate reader. B. Biofilm membrane-forming conditions undergoing different incubation times.
Dry Weight change of the adherent biofilm on K3 carrier packing of MBBR
Saccharomyces cerevisiae BY4741 and BY4741 FLO11+ strains were grown in batches in a shake flask containing 50mL YPD with an initial inoculum OD600 = 0.2, and five K3 fluidized bed fills were added for each group. After 1,2,3 and 4 days of culture in each group, the K3 packing was removed. After naturally drying, the total dry weight change was recorded and the biofilm dry weight change curve was drawn. Three parallel experiments were done for each data point.
Figure 9. K3 fluidized bed carrier packing adhesion experiment.
After 3 days of batch culture, obvious biofilm adhesion (Figure 4-10) appeared on the K3 fluidized bed fills cultured with the BY4741 FLO11+ group strains. The biofilm dry weight change curve shows that the amount of film reached the maximum (Figure 4-11), which may be related to the growth cycle and state of the cell. So the best bioadsorption process can be preliminarily judged.
Figure 10. Strain overexpressing FLO11 could adhere to form significant biofilm on the K3 fluidized bed carrier packing, while no adhesion was observed in the BY4741 strain.
Figure 11. The K3 fluidized bed carrier filler was added at 0 h, Biofilm dry weight change curves on carrier filler after 20 h and 24,48,72 and 96 h of batch culture.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 495
Illegal SpeI site found at 3562
Illegal PstI site found at 4409 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 495
Illegal SpeI site found at 3562
Illegal PstI site found at 4409 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 508
Illegal XhoI site found at 4440 - 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 495
Illegal SpeI site found at 3562
Illegal PstI site found at 4409 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 495
Illegal SpeI site found at 3562
Illegal PstI site found at 4409 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 182
Illegal SapI site found at 1436
Illegal SapI site found at 1472
Illegal SapI site found at 1517
Illegal SapI site found at 1553
Illegal SapI site found at 1589
Illegal SapI site found at 1634
Illegal SapI site found at 1670
| None |