Autolysin

Autolysin

Preparation of Autolysin
1. Toothpick a single colony and grow up in 0.2ml TAP or M medium for 2-3 days until culture becomes moderately green. Transfer the 0.2ml culture to 10ml of TAP medium in a glass tube, grow under bright light for 3-4 days until dark green. Spread 1.5ml of this culture on a large plate of M agar. Make 2 plates for each mating-type strain. Incubate the plates under bright light for 4-5 days until a green lawn of cells is formed on each plate.
2. Add 15ml of M-N medium to each plate, scrape off cells by gently spreading with a bent glass pipette. Transfer cells to a sterile 50ml tube. Wash the plate once with another 10ml of M-N. Combine cells from the two plates for each stain.
3. Pipette 20m l of the cell suspension into 930m l ddH₂0 add 50m l of 10% glutaraldehyde. Count the cell density with a hemacytometer.
4. Centrifuge in IEC centrifuge for 10 minutes at 25000RPM. Pour off the supernatant as quickly as possible after centrifugation is done.
5. Resuspend cells in M-N to a final concentration of 8-10 * 10⁷ cell/ml. Transfer to a sterile 500ml flask and incubate culture under bright light for 3-4 hours.
6. After 2-4 hours, mix a small sample of the two mating-type cultures and assess for mating activity. If very good mating occurs, mix equal volume of the mating cultures in a sterile 1-liter flask. Incubate under bright light.
7. After 30 minutes, fix a small sample of the mating mixture with glutaraldehyde. Estimate the % if quadriflagellates. If % is greater than 80% go to the next step. Or else, repeat steps 1-6.
8. Transfer mating mixture to a sterile 250ml round bottom bottle, centrifuge at 2800 RPM for 15 minutes at 15 degrees C in the IEC centrifuge.
9. Transfer supernatant to another sterile bottle, centrifuge at 10000 RPM at 4 degrees C for 10 minutes un Sorvall with a GSA rotor.
10. Transfer supernatant to a sterile container. Aliquot 5-10ml fractions and store in -80 degrees C.

Autolysin Assay
1. Grow up 200ml of cells in a flask. Count the cell number and transfer 5*10⁷ cells to each 15ml corning tube. Centrifuge at top speed in a clinical table-top centrufuge. Discard supernatant.
2. Thaw autolysin at room temperature. Add 0.5, 1, 2, or 4ml of autolysin to cell pellets. Resuspend cells by gently shaking the tubes. For control, use M-N to resuspend the cells. Incubate at room temperature with gentle shaking for 45 minutes.
3. After incubation, add 50m l of each sample to 50m l of 0.5% glutaraldehyde or 1% NP-40 in a microtiter well. Estimate % of lysis as soon as possible (within 5 minutes after addition to NP-40). For use in transformation, autolysin prep. that gives more than 80% cell lysis will be required.

Chlamydomonas Transformation with Autolysin
1. Pick a single colony of Chlamy cells into 200m of medium in a microtiter well. Grow for 2-3 days until culture turns green. Transfer cells to 10ml medium in a tube, grow cells for 3-4 days until culture turns green. Transfer the 10ml culture to 250ml medium in a bubbler. Grow cells for 3-4 days to 1-5 * 10⁶ cells per ml. (For Arg- strains, use TAP + 50 m g/ml arginine; for Nit- strains, use SGII).
2. For each transformation, aliquot 4 * 10^7 cells in a 15ml corning tube Pellet cells by centrifugation at max. speed in a table-top clinical centrifuge for 3 minutes at RT. Resuspend cells in an appropriate volume of autolysin (determined by autolysin test). (If you have too large a volume to fit in a 15ml tube, use a 50ml tube and transfer cells to a 15ml tube after resuspending them in autolysin).
3. Incubate cells for 50-60 minutes at RT with gentle agitation on a rotatory shaker. Pellet cells by centrifugation for 2-3 minutes in the table-top clinical centrifuge. (If there are more than 2 or 3 transformations to do, set up tubes with time in between so that you will have time to do the next few steps before the next ones are ready).
4. Add 0.3ml of SGII-NO3 and resuspend cells with a sterile glass pipette. Add about 0.3g acid-washed sterile glass beads. Add DNA (1m g of linearized plasmid, supercoiled DNA will also work to a lesser extent).
5. Add 100m l of 20% PEG-8000 (Sigma. filter-sterilized and stored in a wrapped bottle at RT). Immediately, start vortexing cells at top speed for 45 seconds using a Vortex Genie mixer.
6. Immediately add about 0.6ml SGII-NO3 medium to the cells. Transfer all the cells to a large (150mm diameter) agar plate (M for Arg- strain, MNO3 for Nit- strain) with a sterile glass pipette. Spread cells gently on the plate.