iGEM 2019 — Team Tartu_TUIT

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# Team:Tartu TUIT/Notebook

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[Characterization](/Team%3ATartu_TUIT/Characterization)
[Design](/Team%3ATartu_TUIT/Design)

Results

[Results](/Team%3ATartu_TUIT/Results)
[Modeling](/Team%3ATartu_TUIT/Model)
[Demonstration](/Team%3ATartu_TUIT/Demonstration)
[Improve](/Team%3ATartu_TUIT/Improve)

Human Practices

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[Public Engagement](/Team%3ATartu_TUIT/Public_Engagement)
[Collaborations](/Team%3ATartu_TUIT/Collaborations)

Lab

[Notebook](/Team%3ATartu_TUIT/Notebook)
[Experiments](/Team%3ATartu_TUIT/Experiments)
[Safety](/Team%3ATartu_TUIT/Safety)

People

[Attributions](/Team%3ATartu_TUIT/Attributions)
[Team](/Team%3ATartu_TUIT/Team)
[Sponsors](/Team%3ATartu_TUIT/Sponsor)

#

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# Team:Tartu TUIT/Experiments

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Project

[Description](/Team%3ATartu_TUIT/Description)
[Parts](/Team%3ATartu_TUIT/Parts)
[Characterization](/Team%3ATartu_TUIT/Characterization)
[Design](/Team%3ATartu_TUIT/Design)

Results

[Results](/Team%3ATartu_TUIT/Results)
[Modeling](/Team%3ATartu_TUIT/Model)
[Demonstration](/Team%3ATartu_TUIT/Demonstration)
[Improve](/Team%3ATartu_TUIT/Improve)

Human Practices

[Human Practices](/Team%3ATartu_TUIT/Human_Practices)
[Public Engagement](/Team%3ATartu_TUIT/Public_Engagement)
[Collaborations](/Team%3ATartu_TUIT/Collaborations)

Lab

[Notebook](/Team%3ATartu_TUIT/Notebook)
[Experiments](/Team%3ATartu_TUIT/Experiments)
[Safety](/Team%3ATartu_TUIT/Safety)

People

[Attributions](/Team%3ATartu_TUIT/Attributions)
[Team](/Team%3ATartu_TUIT/Team)
[Sponsors](/Team%3ATartu_TUIT/Sponsor)

#

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While doing scientific experiments, it is important to make and follow uniform protocols to eliminate as many variables as possible and to ensure repeatability of the experiment. We followed protocols provided by both our sponsors and supervisors. If some steps in our experiments failed repeatedly, we changed a few parameters of protocols like incubation time or amount of DNA, however, protocols written here were our main guidelines and we tried not to diverge much far from them.

**Phusion DNA polymerase protocol**

To amplify DNA fragments for subsequent cloning, PCR with Phusion DNA polymerase was used. Components of PCR reaction are listed in Table 1.

Table 1. Components of PCR reaction(table below)

| Components | 50 µL reaction | Final concentration |
| --- | --- | --- |
| Nuclease-free water | add to 50 µL | - |
| Buffer 5X (HF or GC) | 10 µL | 1X |
| dNTPs mix (25 mM each) | 0.4 µL | 200 µM each |
| DMSO((CH3)2SO) (Optional) | 1.5-2.5 µL | 3-5% |
| Forward primer (10 µM) | 2.5 µL | 0.5 µM |
| Reverse primer (10 µM) | 2.5 µL | 0.5 µM |
| DNA template: Genomic DNA,Plasmid DNA | X µL; X µL | 50 ng - 250 ng; 1 pg–10 ng |
| Phusion DNA Polymerase | 0.5 μLl | 0.02 U/µL |

All the components of the PCR reaction were mixed on ice. PCR tubes with the reaction mix were transferred into a preheated to 98 oC thermocycler block and the PCR were performed according to the conditions listed in the table 2.

#### Table 2. PCR machine setup(table below)

| Step | Temperature (°C) | Time (s) |
| --- | --- | --- |
| Initial Denaturation | 98 | 30 |
| 25-35 Cycles | 98 | 10 |
| 45-72 (depends on primers’ Tm) | 20 |
| 72 | 15-30 s per kb |
| Final Extension | 72 | 15-30 s per kb |
| Hold | 15 | ∞ |

### Restriction

All the restriction enzymes used in our work were FastDigest enzymes from Thermo Fisher Scientific. Restriction reactions were prepared by mixing the following reagents (Table 1.):

#### Table 1. Restriction reaction mix for 20 μL volume(table below) | Components | Volumes (°C) | | --- | --- | | Water | add to 20 μL | | 10X FD Buffer | 2 μL (1X) | | DNA | 1 μg | | | 15-30 s per kb | | Restriction enzymes | 1 μL | | FastAP (for vector DNA only)\* | 0.5 μL | \*FastAP (Thermosensitive Alkaline Phosphatase) was added to the vector restriction reaction in order to prevent self-ligation of the vector fragments. All the components of restriction reactions were mixed in a 1.5 mL tube. After mixing, the restriction reaction was incubated at 37°C for 30-60 min. Following the incubation, restriction enzymes were inactivated by heating the mixture for 15 min at 65-85°C. At the next step, PCR clean-up protocol or gel electrophoresis followed by DNA purification from the gel was performed to clean the restricted DNA fragments. Agarose gel for DNA separation 1. In order to make 1% agarose gel, 1 g of agarose was added to 100 mL of TAE [tris(hydroxymethyl)aminomethane - ethanoic acid - ethylenediaminetetraacetic acid] buffer solution. 2. The mixture was heated in a microwave until agarose was completely melted. 3. The mix was left to cool down to approximately 60°C. 4. 6 μL of Atlas ClearSight DNA Stain was added. 5. The solution was poured into a gel caster with an appropriate comb installed. 6. After polymerization, the gel was used for DNA electrophoresis. DNA purification from agarose gel DNA was purified from the gel using the Gel-Out kit from A&A Biotechnology according to the manual instructions. A brief description of the procedure is written below. 1. DNA fragments of the correct sizes were cut out from the gel and transferred to Eppendorf tubes. 2. R7SI agarose melting solution was added; in case of < 2% agarose gel - 400 μL, in case of ≥ 2% agarose gel - 500 μL. Samples were incubated at 50°C until the complete dissolution of agarose slices. During incubation, the samples were mixed from time to time by inverting the tubes or vortexing. 3. An appropriate volume of Isopropanol was added. 4. < 2% agarose gel - 200 μL. 5. ≥ 2% agarose gel - 250 μL. 6. The samples were mixed by inverting the tubes. 7. After brief centrifugation, the mixture was transferred onto the minicolumns, which were centrifuged for 30 s at 10 000-15 000 RPM. 8. The flow-through was discarded and the minicolumns were re-assembled with the collection tubes. 9. At the next step, 600 μL of A1 wash solution were added and centrifugation for 30 s at 10 000-15 000 RPM was performed. 10. The filtrates were discarded and minicolumns were re-assembled with the collection tubes. 11. 300 μL of A1 wash solution were added. 12. Centrifugation for 1 min at 10 000-15 000 RPM was performed. 13. The dry minicolumns were transferred to the new 1.5 mL elution tubes and 50 μL of TE buffer were added directly onto the minicolumns resin. 14. Minicolumns were incubated for 3 min at room temperature and centrifuged for 60 s at 10 000-15 000 RPM. 15. The minicolumns were discarded. Purified DNA was used right away or kept at -20°C until needed. DNA concentration measurements DNA concentration measurements were performed with NanoDrop Microvolume Spectrophotometer according to the manufacturer manuals. Software used: NanoDrop 1000 v.3.8.1. Mode used: Nucleis Acid, DNA-50 Ligation All ligation reactions were performed using T4 DNA ligase from NEB according to the manufacturer's instructions. All the components were mixed in Eppendorf tube in accordance with Table 1. DNA was taken using the 1:3 ratio of vector:insert based on the size of DNA fragments. Table 1. Components of the ligation reaction (table below) | Components | Amount | | --- | --- | | Water | add to 20 μL | | 10X FD Buffer | 2 μL (1X) | | Vector | 0.020 pmol | | Insert | 0.060 pmol | | T4 DNA Ligase | 1 unit (5 units for blunt-end ligation) | | PEG 4000 (for blunt-end ligation, optional) | 2 μL | Ligation reactions were kept at 16°C for 2-4 hours or overnight (if ligation contains blunt ends). 1-2 µL of ligation mixture were used for bacterial transformation. Bacterial transformation DNA transformation to bacteria cells was performed as follows: 1. *Escherichia coli* Dh5α we transferred from -80°C freezer to ice. E.coli cells were left on ice for approx. 10 min to melt. 2. 50 μL of cells (DH5α) was mixed with 2 μL of ligation reaction (50 pg - 1 ng for plasmid DNA). 3. Cells were left for 30 min on ice. 4. Heat-shock was performed at 42°C for 30 s. 5. Cells were cooled for 1-2 min on ice. 6. 1 mL of LB medium was added to the cells. 7. Tubes were incubated in the shaker at 37°C and 220 rpm for 40 min. 8. Meanwhile, bacterial selection plates (LB with antibiotics) were transferred from the cold room to room temperature (Plates were labeled accordingly and a few pieces of glass beads were added to each of them). 9. After incubation, Eppendorf tubes with bacteria were centrifuged for 1 min at 5000 rpm. 10. Excess of supernatant was discarded (approx. 250 µL were left in the tube). 11. Bacterial cells were resuspended in the supernatant leftover and transferred on the selection plate. 12. Bacterial cells were spread on the plate surface evenly using sterile glass beads. 13. Glass beads were removed and plates were incubated at 37°C for 12-14 hrs (until single colonies appeared). Plasmid extraction Plasmids were extracted from bacterial cells using the “Plasmid Mini” kit by A&A Biotechnology according to manual instructions. A brief description of the procedure is written below. 1. Up to 3 mL (1.5-3 mL) of overnight bacterial culture was centrifuged. After that supernatant was removed and the bacterial pellets were suspended in 200 µL of L1 cell suspension solution. 2. 200 µL of L2 lysis solution was added and the solution was carefully mixed by turning the Eppendorf tube up-side-down. The mix was incubated for 3 min at room temperature. 3. 400 µL of GL3 neutralizing solution was added and carefully mixed until the disappearance of the raspberry color of the lysates. 4. The lysates were centrifuged for 10 min at 10 000-15 000 RPM. 5. The supernatants were transferred onto the minicolumns, which were centrifuged for 1 min at 10 000-15 000 RPM. 6. The minicolumns were removed from the collection tubes and the filtrates were discarded then the minicolumns were re-assembled with the same collection tubes. 7. 500 µL of W first wash solution were added and the minicolumns were centrifuged for 1 min at 10 000-15 000 RPM. 8. The minicolumns were removed from the collection tubes, the filtrates were discarded and the minicolumns re-assembled with the same collection tubes. 9. 600 µL of A1 second wash solution were added and centrifugation for 2 min at 10 000-15 000 RPM was performed. 10. The dry minicolumns were carefully transferred into new 1.5 mL tubes. 60 µL of TE buffer was added directly onto the minicolumns resin to elute DNA. 11. Minicolumns were incubated for 3 min at room temperature followed by 1 min centrifugation at 12 000 RPM. 12. Eluted plasmid DNA was used straight away or stored at -20°C until needed. Yeast transformation In order to genetically transform yeast cells, the desired yeast strain was pre-grown in 50 mL of liquid YPD (yeast extract peptone dextrose) until OD600 0.6-0.8. The genetic transformation was performed based on the following protocol: 1. Cell culture was centrifuged in a sterile 50 mL falcon tube. The supernatant was discarded. 2. The cell pellet was resuspended in 1 mL of sterile 100 mM LiAc(Lithium Acetate) in TE (tris(hydroxymethyl)aminomethane - Ethylenediaminetetraacetic acid buffer) and transferred into the Eppendorf tube. 3. Cells were centrifuged at 3600 rpm for 60 s. The supernatant was removed and cells were resuspended again in two times of cell volume of 100mM LiAc in TE. 4. Cells were incubated for 10 min at room temperature (competent cells). 5. Single-stranded carrier DNA was prepared: Salmon Sperm DNA(SS-DNA) was boiled for 10 min at 100°C and immediately chilled on ice before use. This ensured that the DNA was single-stranded. SS-DNA was kept on ice for the rest of the time before use. 6. 20 μL of plasmid DNA (yeast integration vector restricted with proper restriction enzyme) and 10 μL of SS-DNA was mixed in a separate 1.5 mL tube. 7. 100 μL of yeast competent cells were added to the DNA and mixed. 8. At the next step, 700 ul of sterile PEG(Poly(ethylene glycol))/LiAc (40% PEG 3350 + 100mM LiAc in 1 x TE) were added. 9. Finally, 48 μL of DMSO((CH3)2SO) was added and everything was gently mixed by pipetting. 10. The mixture was incubated at 42°C for 40 min (heat shock). 11. After the heat shock, the cells were chilled on ice for 2 min. 12. At the next step, the cells were centrifuged for 30 s at 6000 rpm at room temperature. The supernatant was removed and the cells were resuspended in 1 mL of sterile 1x TE buffer. 13. After that, the cells were centrifuged for 60 s at 3600 rpm at room temperature. The supernatant was discarded and the cells were resuspended in 200 μL of sterile 1x TE buffer. 14. Finally, 200 μL of suspension were transferred on the yeast selection plates and cells were evenly spread all over the plate surface using sterile glass beads. 15. The plates were incubated upside down at 30°C until colonies appeared (usually, for 2-3 days). Yeast colony PCR Preparation of yeast cell lysates 1. Cells of the single yeast colony (at the end of the pipette tip) were transferred into 30 µL of 20 mM NaOH in the Eppendorf tube. 2. The mixture was boiled at 100°C for 10 min. 3. Lysates were placed on ice for cooling followed by 1 min centrifugation at 12000 rpm. 4. Yeast cell lysates were placed on ice. 5. 1 µL of supernatant was used as a template for PCR reaction. Preparation of PCR mixture Components of the PCR reaction were mixed according to Table 1. Table 1. Components for PCR with Taq polymerase(table below) | Components | 20 μL Reaction | | --- | --- | | Water | till 20 μL | | dNTPs (25 mM each) | 0.25 μL | | Primers (10 μM) | 0.5 μL each | | MgCl2 (50 mM) | 1 μL | | 10xYPB Buffer (Solis BioDyne) | 2 μL | | Taq polymerase (Solis BioDyne) | 0.3 μL | | DNA template (cell lysate) | 1 μL | Conditions used for yeast colony PCR are described in Table 2. Table 2. PCR machine setup(table below) | Step | Temp | Time | | --- | --- | --- | | Initial Denaturation | 95o C | 5 min | | 25-35 Cycles | 98 (Denaturation) | 30 s | | 48 o - 60 o C (Primer annealing) | 20-35 s | | 72 o C (Elongation) | 1 min/kb | | Final Extension | 72o C | 5 min | | Hold | 15o C | | **LB(Lysogeny broth) and YPD media** Table 1. LB medium components(table below) | | | | --- | --- | | Tryptone | 10 g | | Yeast extract | 5 g | | NaCl | 10 g | | H2O up to | 1 L | Table 2. YPD components(table below) | | | | --- | --- | | Peptone(Formedium) | 20 g/L | | D-Glucose anhydrous | 20 g/L | | Micro Granulated Yeast Extract | 10 g/L | | Bacto agar(for plates) | 15 g/L | 1. All the components were mixed in 2 L Erlenmeyer flask with a magnetic stirrer in it. 2. 15 g of Bacto Agar (for plates only, Bacto Agar was omitted for liquid medium) were added. 3. The medium was autoclaved for 15 min at 121°C. 4. The medium was left to cool down to approximately 55°C. 5. 1 mL of sterile 100 mg/mL antibiotic solution (penicillin) was added to the medium and mixed. 6. Approx. 30 ml of medium was poured into the sterile Petri dishes and was left for cooling down and polymerization. 7. Plates were kept at +4°C for up to two weeks. **Microscopy** For the promoter characterization part, yeast colonies were checked after the transformation for the presence of GFP fluorescence. Prior to the experiment, yeast cultures were grown in CSM uracil dropout medium with 2% glucose as a carbon source. The optical density (OD600) was in the range 0.2-0.6. Cultures were observed under a Zeiss Axio Observer Z1 microscope. The following settings were used for the microscope: 25 ms exposure time for phase contrast channel, 100 ms exposure time for EGFP channel. Colibri LED modules were used as a source of 470 nm excitation wavelength at 25% intensity. **Time-lapse microscopy** Prior to time-lapse microscopy, the yeast cells were grown in synthetic complete (SC) media lacking histidine or SC lacking histidine and tryptophan depending on the integrative plasmids in the strain. The cultures were grown to OD600 0.2-0.8, the cells were pipetted onto a 0.08 mm cover glass slip and were covered with 1.5% agar-SC. For induction of the estradiol-controlled promoter, 1 μM estradiol was added to the SC-agar. Zeiss Observer Z1 microscope with 63C/1.4NA oil immersion objective and Axiocam 506 mono camera was used for imaging. The focus was kept using Definite Focus and the sample was kept at 30 °C using PeCon TempControl 37-2 digital. The cells were imaged every 5 minutes using brightfield illumination and the experiments were 13-18 hours long. **Plate reader** Plate readers are instruments that are used to detect samples in microtiter plates. This year as a team has used BioTek Synergy MX microplate reader to measure the cells’ optical density and intensity of EGFP fluorescence. All strains used in the experiments were pre-grown overnight at 30°C in liquid CSM/2%Glc media and diluted to OD600 in a range of 0.9 to 1.1. Then diluted cell cultures were distributed to 96 well plates (clear flat bottom).4 replicates of 2 colonies from each strain were used. 200 µL of cells were added to each well. CSM/2%Glc media was used as a reference to OD600. The measurements were done with the following parameters. Absorbance was measured at 600 nm and when it comes to fluorescence it was measured at these conditions -excitation 485 nm, emission 528 nm, bandwidth 20, gain 80. **Western blotting** 1. The yeast strains were pre-grown in 10ml CSM media overnight in the 300C shaker. 2. Before the induction of Glucanase expression (Timepoint 0), 5ml of cell culture was collected (centrifugation at 4000 rpm for 1 minute), proteins in the supernatant were concentrated using Acetone precipitation method. 3. Another half of the cell culture was induced with Beta-estradiol (final concentration 1μM) and incubated for 6 hours. 4. After 6 hours incubation, cells and supernatant were collected as described in 2. 5. Cell pellets were resuspended in 200 µl of Urea buffer, 250 µl of glass beads were added. 6. Cells were disrupted using BeadBeater(4 m/s for 40s) 7. The samples were centrifuged for 10 min at 14200 rpm. 8. Protein precipitates from the supernatant were resuspended in 200 µl of Urea buffer. 9. Samples were loaded to the SDS-PAGE. 10. SDS-PAGE (Denaturing Poly acrylamide gel electrophoresis) was performed using the following parameters: 15 mA, 300V 1h 20 min. 11. To transfer proteins from the gel onto a membrane the semi-dry western blot transfer method was used: 60 mins transfer at 25V and 1A. 12. The nitrocellulose membrane has high affinity for proteins and antibodies and therefore its necessary to minimize the nonspecific antibody binding. The membrane is blocked in 5% dry milk, 1X TBS-T buffer solution, and incubated with agitation for 1 hour. 13. After blocking, the membrane is then incubated with agitation for 1 hour in solution containing blocking solution and 1:1500 dilution of primary antibody (Anti-HA) that specifically recognizes protein of interest. 14. This is followed by washing with 1X TBS-T buffer for 15 mins and 3 more times for 5 mins with fresh buffer under mild agitation to remove nonspecific binding primary antibody. 15. The membrane was then incubated with secondary antibody (1:7500 dilution rabbit anti-mouse in 3% dry milk, 1X TBS-T buffer solution) with agitation for 30 mins and then washed with 1X TBS-T buffer to remove excess secondary antibody: 1 time for 15 minutes and 3 times for 5 minutes in quick stirs. 16. Result manifestation using Supersignal West Pico Rabbit IgG Detection Kit. Exposure time used ranged from 10 seconds to 10 minutes. **Concentration of the proteins from the supernatant** **Acetone precipitation of proteins** **Materials Required** • Cold (-20°C) acetone, a volume four times that of the protein samples to be precipitated • Centrifuge tube, made of acetone-compatible polypropylene and able to hold five times the sample volume • Centrifuge and rotor for the tubes used, minimum 13,000 × g required **Protocol** 1. Cool the required volume of acetone to -20°C. 2. Place the protein sample in an acetone-compatible tube. 3. Add four times the sample volume of cold (-20°C) acetone to the tube. 4. Vortex tube and incubate for 60 minutes at -20°C. 5. Centrifuge 10 minutes at 13,000-15,000 × g. 6. Decant and properly dispose of the supernatant, being careful to not dislodge the protein pellet. Optional: If additional cycles of precipitation are necessary to completely remove the interfering substance, then repeat steps 2-5 before proceeding to step 7. 7. Allow the acetone to evaporate from the uncapped tube at room temperature for 30 minutes. Do not over-dry pellet, or it may not dissolve properly. 8. Add buffer appropriate for the downstream process and vortex thoroughly to dissolve the protein pellet. [Acetone precipitation of proteins](http://tools.thermofisher.com/content/sfs/brochures/TR0049-Acetone-precipitation.pdf) **Amicon Ultra-15 Centrifugal filters** 1. Add up to 15 mL of sample (12 mL if using a fixed-angle rotor) to the Amicon® Ultra filter device. 2. Place capped filter device into centrifuge rotor; counterbalance with a similar device. 3. When using a swinging-bucket rotor, spin the device at 4,000 × g maximum for approximately 15–60 minutes. When using a fixed-angle rotor, orient the device with the membrane panel facing up and spin at 5,000 × g maximum for approximately 15–60 minutes. 4. To recover the concentrated solute, insert a pipettor into the bottom of the filter device and withdraw the sample using a side-to-side sweeping motion to ensure total recovery. The ultrafiltrate can be stored in the centrifuge tube. NOTE: For optimal recovery, remove concentrated sample immediately after centrifugation. [Amicon Ultra-15 Centrifugal filters](http://www.merckmillipore.com/INTL/en/product/Amicon-Ultra-15-Centrifugal-Filter-Units%2CMM_NF-C7715#documentation) **HPLC** Column used: BioRad Aminex HPX-87 H. Mobile phase - 0.005M H2SO4. Flask preparation: 1. Grow S.cerevisiae W303 wild-type strain in 5 ml YPD in 15ml tube overnight. 2. Change the media to 5ml 1% glucose minimal Delft medium. Keep cells for 6 hours. 3. To prepare 125ml flask with 20ml of 1% glucose minimal Delft medium and culture with initial OD600 ~0.1. Make triplicates. 4. Check OD600 and collect 500 µl of culture for the HPLC sample every 2 hours during the exponential phase and take one point in 24 hours. Sample preparation for HPLC: 1. Centrifuge collected samples with 5500rm for 5 min. 2. Transfer the supernatant to another Eppendorf tube. 3.Centrifuge with maximum speed for 5 min. 4. Transfer the supernatant to another Eppendorf tube. 5. Prepare glass tubes with 200 µl of the sample. 6. Run each sample three times. **Spot viability assay** 1. Cells were grown for 12 hours at 30°C 160 rpm with and without the induction. 1 µM Estradiol was used. 2. Cultures were diluted to the concentration of 10^6 cells/ml (0.08 OD) in YPD media, YPD with 10% DMSO, to make membrane permeable, and in water, to create hypo-osmotic stress conditions. 3. Serial dilutions were made(1, ¼, 1/20, 1/200) 4. 5 µl of the dilutions were plated on the YPD plates. 5. Cells were grown for 24 hours at 30°C.

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# Team:Tartu TUIT/Results

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Project

[Description](/Team%3ATartu_TUIT/Description)
[Parts](/Team%3ATartu_TUIT/Parts)
[Characterization](/Team%3ATartu_TUIT/Characterization)
[Design](/Team%3ATartu_TUIT/Design)

Results

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**Time-lapse Microscopy Shows Autolysis of Yeast Cells**

We have tested six strains in time-lapse microscopy experiments following the cells for up to 20 hours after induction of glucanase genes from the estradiol-activated promoter. The strains were expressing the following glucanases: yeast Scw4 and bacterial enzymes Bba, α-Bba, Ost1-Bba, α-Glc1 and Ost1-Glc1.

The experiments revealed that an increase in cell size could be detected at around 5 hours after the start of glucanase induction. The origin of the slow responsiveness could be that the estradiol induction in general shows relatively slow target protein accumulation dynamics [[1]](#ref-1). The overexpression of the yeast glucanase Scw4 had a minor effect on cell morphology, however, it did induce lysis of very few cells (**Fig. 1A**). A more defined effect can be seen in the strains that express bacterial genes (**Fig. 1B-F**). In the presence of estradiol these cells have abnormal morphology: the cells are enlarged, have swelled vacuoles, some are elongated, possibly due to cell wall defects.

In the later stages of the experiment, at around 8 hours from the start of the induction, cells start dying rapidly. There were cells with defined lysis, in which case the release of intracellular material could be detected (see **Movies 1** and **2**). Regrettably, we were unable to get all cells lysed even after 20 hours of induction. The most probable explanation is that the secretion signals we used were not very effective. 20 hours after the start of glucanase induction, about 7-13% of cells had died, whereas the percentage of dead cells was around 1% or less in the positions without estradiol (**Fig. 2**, **Table 1**). No significant difference was noticed between the four strains with the combinations of bacterial genes and secretion signals. However, the addition of secretion signals to Bba has enhanced its activity, as cells expressing Bba fused with secretion signals grew at a lower rate and the percentage of dead cells by the end of the experiment was increased (**Fig. 2**).

[](https://static.igem.org/mediawiki/2019/6/67/T--Tartu_TUIT--Ost1-Glc1_new.mp4)
**Movie 1.** The effect of *Ost1-GLC1* expression was followed with time-lapse microscopy. On the left uninduced cells, on the right *Ost1-GLC1* expressing cells.

[](https://static.igem.org/mediawiki/2019/8/8e/T--Tartu_TUIT--Ost1-BBa_new.mp4)
**Movie 2.** The effect of *Ost1*-BBa\_K2711000 expression was followed with time-lapse microscopy. On the left uninduced cells, on the right *Ost1*-BBa\_K2711000 expressing cells.

Interestingly, when one cell is lysed, the surrounding cells often lyse as well. This can be explained by a large amount of glucanases being released at once. This effect can be valuable as every lysed cell will speed up the process. Although the lysis efficiency is small, the time-lapse microscopy experiments show a proof of concept that controlled yeast self-lysis can be induced by expression of glucanases.

![](https://static.igem.org/mediawiki/2019/7/7b/T--Tartu_TUIT--Results1.png)
![](https://static.igem.org/mediawiki/2019/7/77/T--Tartu_TUIT--Results2.png)
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![](https://static.igem.org/mediawiki/2019/2/24/T--Tartu_TUIT--Results6.png)
**Figure 1. Time-lapse microscopy of six yeast strains grown in glucanase expression non-inductive (- panel, without ß-estradiol) or inductive (+ panel, with ß-estradiol) conditions.** Brightfield (BF), GFP fluorescence and merged channel images are shown. Minutes indicate the time from ß-estradiol addition. Different strains contain different glucanase genes expressed from LexA promoter and are represented in following order: (A) SCW4; (B) BBa\_K2711000; (C) α-factor pre-pro signal fused to BBa\_K2711000; (D) Pre-Ost1-pro-α-factor signal fused to BBa\_K2711000; (E) α-factor pre-pro-Glc1 fusion; (F) Pre-Ost1-pro-α-factor-Glc1 fusion.

![](https://static.igem.org/mediawiki/2019/6/60/T--Tartu_TUIT--Results7.png)
**Figure 2. The effect of glucanase expression on cell viability.** The percentage of dead cells was counted from time-lapse microscopy experiments 20 hours after the start of experiment. The experiment was performed with six strains expressing different glucanases from the estradiol-responsive promoter. The plot shows the percentage of dead cells in the presence or absence of estradiol in the medium. For the number of cells used in the analysis, see Table 1.

**Table 1.** The number of cells counted for each condition in the calculation of the percentage of dead cells presented in Figure 2.

|  | Number of cells analyzed | |
| --- | --- | --- |
| Glucanase | Uninduced culture | Induced culture |
| α-Glc1 | 2587 | 1518 |
| Ost1-Glc1 | 2355 | 1797 |
| BBa | 1097 | 379 |
| α-BBa | 1504 | 1383 |
| Ost1-BBa | 589 | 453 |
| SCW4 | 1120 | 873 |

**Autolysis Is Inefficient in Liquid Cultures**

To test the effects of glucanase expression on yeast cells in liquid cultures, the cells were grown in synthetic complete media with or without of β-estradiol. We included four strains in the assay: the background strain NS202, that expresses only the estradiol-responsive transcription factor, and three strains that contain glucanase genes (Scw4, Ost1-Glc1 and Ost1-Bba) under the control of the estradiol-inducible promoter. In the presence of β-estradiol, the glucanases Scw4, Ost1-Glc1 and Ost1-Bba are induced. The induction led to a decreased growth rate and slightly enlarged cell size; however, no lysis of cells could be detected by measuring the total protein concentration in the media using Bradford assay (data not shown). This indicates that sufficient extracellular levels of the glucanases to induce cell lysis are not reached in liquid cultures.

To test if the increased cell size phenotype detected in the cultures with glucanase expression is accompanied by a weakened cell wall, we tested the resistance of these cells to hypo-osmotic stress and to the presence of DMSO (**Fig. 3**). The cultures were diluted either in water, to induce hypo-osmotic stress or in YPD with 10% DMSO. DMSO increases membrane permeability, leading to a hypothesis that DMSO might promote lysis of cells with weakened cell walls. However, we did not see significant differences in cell viability in the tested stress conditions (**Fig. 3**). We note a decrease in the growth rate of cells diluted in H2O, but as this is similar for all tested strains and conditions, this is presumably due to cell cycle arrest caused by nutrient depletion.
We presume that the inability to achieve cell lysis by glucanase induction in liquid cultures is due to low secretion efficiency. We detected some lysis in cells grown on agar pads (**Fig. 1**), suggesting that when cells are grown on agar, the local concentration of the secreted enzymes increases around the cells due to limited diffusion. In liquid cultures, however, the enzymes are diluted, and the lysis is inefficient.

![](https://static.igem.org/mediawiki/2019/a/a4/T--Tartu_TUIT--Results8.png)
**Figure 3. Viability assay to test the effect of glucosidase expression on resistance to hypo-osmotic stress and DMSO.** Yeasts were grown to stationary phase in the absence or presence of 1 µM β-estradiol, which induces the expression of glucanases Scw4, Ost1-Glc1, and Ost1-Bba. The cultures were first diluted to 106 cells/ml either in YPD, H2O or YPD with 10% DMSO, followed by serial dilutions of 4x, 20x and 200x. The dilutions series were plated on YPD plates and grown for 24 hours to estimate the effect of stress conditions on cell viability.

**Western Blotting Confirms Low Secretion Efficiency**

We used Western blotting to check whether the target proteins are expressed in yeast cells after induction of the synthetic promoter with β-estradiol and to test the efficiency of their secretion. For this, we created strains expressing HA-tagged versions of Glc1 fused to different secretion signals (Ost1 or α-factor). Prior to addition of estradiol to the medium, no expression of Glc1 could be detected, whereas 6 hours after the induction high concentration of Glc1 had accumulated within the cells (**Fig. 4**). This confirms that the inducible expression is efficient and controllable. We note that Ost1-Glc1 is expressed at significantly higher levels compared to α-Glc1. However, no Glc1 was detected in the supernatant of the centrifuged yeast cell culture (data not shown), although several methods such as acetone precipitation of proteins, immunoprecipitation of 3HA-Glc1 and ultrafiltration were used to concentrate HA-tagged Glc1 from supernatant. This shows that the extracellular concertation of HA-tagged Glc1 protein was below the detection limit, presumably due to inefficient secretion of the Glc1 protein even with the added yeast secretion signals.

Surprisingly, both full-length secretion signal-Glc1 fusion and Glc1 with cleaved secretion signal were detected in cell lysates (**Fig. 4**). The pre-pro secretion signals are cleaved off in two stages: first, the 19 (α) or 22 (Ost1) amino acid pre signal is cleaved in ER, later, the 66 amino acid pro-signal is cleaved in Golgi [[2]](#ref-2). Based on the relatively small difference in electrophoretic mobility, the shorter detected Glc1 fragment likely still contains the pro-signal, indicating that the protein accumulates in the secretory pathway. It has been noted before that overexpression of recombinant proteins can lead to overloading the secretion machinery and specific strains have been designed to overcome this problem [[3]](#ref-3).

![](https://static.igem.org/mediawiki/2019/3/33/T--Tartu_TUIT--WesternBlot.png?fbclid=IwAR31nTY0BLomz11PbciwDolEHr24oUGjHBWKKKoSB2jDG2ZCRJqOMGNoLDQ)
**Figure 4. Western Blot analysis to test the expression and secretion of Glc1.** Western blot image showing the expression of α-3HA-Glc1 and Ost1-3HA-Glc1. The image shows the amount of the protein in cell lysate. The lysate was diluted 20 and 40 times to visualize the two fragments and to avoid signal leak to neighboring lanes.

**GFP Leakage Assay To Measure Cell Lysis**

To evaluate the efficiency of cell wall lysis after glucanase overexpression, we introduced *pTDH3-EGFP* cassette into four strains carrying different glucanase variants. It was expected that if induction of glucanase expression by addition of ß-estradiol leads to cell wall degradation, we might see an increase in the GFP fluorescence in the cell culture supernatant. This method was suggested by experts outside the team and was thought to be more sensitive than the Bradford assay used above.

*Ost1-GLC1*, BBA, *Ost1*-BBA and *α*-BBA carrying strains were pre-grown overnight in liquid cultures and then diluted to the same optical density with fresh medium with (inductive condition) or without (non-inductive conditions) 1 μM β-estradiol. After 8 hours of induction, GFP fluorescence was measured in liquid cultures, cells resuspended in the fresh medium after centrifugation and cell culture supernatant. All measurements were performed with the BioTek Synergy MX microplate reader.

Unfortunately, we did not observe any statistically significant differences in GFP fluorescence in the yeast culture supernatant between induced and non-induced cultures (data not shown). The reason behind this might be very low lysis efficiency, leading to very little release of GFP to the medium, which is under the detection limit of the fluorescence reader. After induction with estradiol, the glucanases are secreted outside of the cells and are diluted by the medium. In this case, the enzymes’ concentration in cell wall proximity does not reach the threshold necessary for cell wall hydrolysis. Evidence for this assumption comes from time-lapse microscopy experiments. When yeast cells are grown on agar pads, glucanases, secreted outside the cells, stay next to the cell wall due to limited diffusion, resulting in higher local concentration and activity. This led to the development of cell wall disruption phenotype (significantly enlarged or popped-out cells) in induced cells (*Ost1-GLC1* and *Ost1*-BBa strains).

**Future Plans**

There are several aspects that can be improved in our project.

To begin with, LexA-ER-AD expression system has proved to be not the most suitable for this purpose, since the response is relatively slow and therefore it does not allow a quick outburst of the enzymes. Additionally, we found that estradiol also affects the growth of cells that do not contain the LexA promoter. This is a toxic of the expression system and can lead to misinterpretation of some results. One possibility is to use light inducible promoters, as these are not only faster but also easier to manipulate and more convenient for a large-scale production.

Another improvement that can be made is experimenting with other yeast secretion signals and other S. cerevisiae strains, which are optimized for protein secretion [[3]](#ref-3). Western blotting revealed that in our strains, the enzymes were still mostly kept inside the cell and potentially had accumulated in ER or Golgi. The secretory efficiency of different recombinant proteins cannot be predicted, as it depends on the folding rate of the protein [[4]](#ref-4). To overcome this limitation, a screening system that connects secretion of the protein of interest to cell growth has been developed [[5]](#ref-5). This system enables identification of optimal secretion signals and could be used to improve the secretion of bacterial glucanases in yeast. Another possibility is to directly target the glucanases to the cell wall using GPI anchors [[6]](#ref-6), however, as for this the glucanase has to be fused with a GPI anchor, it might have a negative effect on the enzymatic activity.

Also, Zymolyase, commonly used for enzymatic dissolution of yeast cell wall, contains protease activity in addition to glucanase activity. The protease activity is needed to degrade the mannoproteins in the cell wall [[7]](#ref-7). Induction of these protease genes in addition to glucanases might further improve the autolysis process.

In addition, some knockouts can be made to weaken cell response to the cell wall stress. Ideally, these should be conditional knockouts that are coregulated with the induced expression of glucanases. Some potential targets could be the genes from HOG (High Osmolarity Glycerol response) pathway or CWI (Cell Wall Integrity) signaling pathway, since these pathways have been previously reported to be responsible for the response to the cell wall stress caused by Zymolyase [[8]](#ref-8) [[9]](#ref-9).

Another possibility is to knock out the genes responsible for the synthesis of other cell wall components, as there is always a risk that cell will adapt to the stressful conditions via enhancing the production of other ones, or β-1,6-Glucan itself. Some examples are chitin synthases: Chs1, Chs2, Chs3 and β-1,3-Glucan synthases: Fks1, Gsc2 [[10]](#ref-10) [[11]](#ref-11).

We can also continue trying out the deletion of genes responsible for assembly and cross-linking, such as *GAS1* (β -1,3-Glucanosyltransferase) [[8]](#ref-8) [[10]](#ref-10) [[11]](#ref-11).

### References:

1. 1. Ottoz DSM, Rudolf F, Stelling J (2014) Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae. Nucleic Acids Res 42:e130. https://doi.org/10.1093/nar/gku616
2. 2. Barrero JJ, Casler JC, Valero F, et al (2018) An improved secretion signal enhances the secretion of model proteins from Pichia pastoris. Microb Cell Fact 17:. https://doi.org/10.1186/s12934-018-1009-5
3. 3. Huang M, Wang G, Qin J, et al (2018) Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production. Proc Natl Acad Sci U S A 115:E11025–E11032. https://doi.org/10.1073/pnas.1809921115
4. 4. Young CL, Robinson AS (2014) Protein folding and secretion: mechanistic insights advancing recombinant protein production in S. cerevisiae. Curr Opin Biotechnol 30:168–77. https://doi.org/10.1016/j.copbio.2014.06.018
5. 5. Bae JH, Sung BH, Kim HJ, et al (2015) An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast. Sci Rep 5:. https://doi.org/10.1038/srep12229
6. 6. De Sampaïo G, Bourdineaud JP, Lauquin GJ (1999) A constitutive role for GPI anchors in Saccharomyces cerevisiae: cell wall targeting. Mol Microbiol 34:247–56. https://doi.org/10.1046/j.1365-2958.1999.01585.x
7. 7. Zlotnik H, Fernandez MP, Bowers B, Cabib E (1984) Saccharomyces cerevisiae Mannoproteins Form an External Cell Wall Layer That Determines Wall Porosity
8. 8. García R, Botet J, Rodríguez-Peña JM, et al (2015) Genomic profiling of fungal cell wall-interfering compounds: Identification of a common gene signature. BMC Genomics 16:. https://doi.org/10.1186/s12864-015-1879-4
9. 9. Levin DE (2005) Cell Wall Integrity Signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 69:262–291. https://doi.org/10.1128/mmbr.69.2.262-291.2005
10. 10. Teparić R, Mrsa V (2013) Proteins involved in building, maintaining and remodeling of yeast cell walls. Curr Genet 59:171–85. https://doi.org/10.1007/s00294-013-0403-0
11. 11. Lesage G, Bussey H (2006) Cell Wall Assembly in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 70:317–343. https://doi.org/10.1128/mmbr.00038-05

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