We designed our Super Safety Strain of S. cerevisiae yeast with industrial scale-up in mind. For engineered strains whose purpose is to synthesize small molecules, such as biofuels and drugs, adequate safeguards are necessary to prevent accidental release and potential production of contaminating small molecules in ecosystems. With that in mind, we had several criteria in mind for our strain:

1. The strain should be incapable of dividing outside of the production facility
2. During production, the strain should be capable of good growth to ensure economic yields of small molecules
3. The strain should be incapable of reverting to a wildtype phenotype capable of growth outside of the production facility
4. The phenotype of the Super Safety Strain should be readily identifiable when the safety system is active

To these ends, we have set out to address these challenges by designing our genetic platform around strains that can switch off expression of telomerase, an enzyme that is critical for maintaining the ends of chromosomes (telomeres), along with a battery of additional mutations to shorten or lengthen the lifespan of the cells.






There are several subunits of yeast telomerase that, when deleted, result in a non-functional telomerase. We chose to utilize the well characterized gene EST2 (Ever Shortening Telomeres 2) which codes for the catalytic subunit of yeast telomerase. The EST2 gene's main function is as a telomerase reverse transcriptase, and EST2 plays an essential role in telomerase activity. The EST2 gene causes telomere extension, and in addition, EST stands for Even Shorter Telomeres. A mutation, such as a deletion, in the EST2 gene will cause the telomeres to shorten and eventually die off.



However, yeast cells also possess a backup system called ALT (Alternative Lengthening of Telomeres) based on telomere extension via homologous recombination. Several genes are required for this pathway to function, There have been many strain backgrounds generated containing EST2 and RAD52 knockouts, with no strains exhibiting "rescue" from senescence.

The RAD52 gene is a DNA recombinase that allows for cells with shortened telomeres to use homologous recombination to re-extend their telomeres. It is involved in repairing DNA double strand breaks by working with RAD51. In cells with a single EST2 deletion, the RAD52 gene codes for an alternate pathway of recovery. In cells with a double deletion for both EST2 and RAD52, they will reach senescence and not recover.

There are certain genes within S. cerevisiae yeast that have been tested with genome-wide screens for deletion mutants that affect telomere length. Two such genes are MAK31 and VPS75. MAK31, generally categorized as a gene involved in protein modification, specifically with N-acetyltransferase, was found by Askree et al. to lengthen the yeast telomeres by 50-150 base pairs. VPS75, a gene that normally deals with vesicular traffic and vacuolar sorting proteins, was found in the same study to shorten yeast telomeres by 50-150 base pairs.

In cells with either a double or triple deletion of MAK31 or VPS75 in conjunction with EST2 and/or RAD52, growth curves should show shifts in when senescence occurs.

W303 diploid and BY4741a wildtype strains were generously donated by the Laboratory of Dr. David A. Sinclair, Harvard Medical School. The W303 diploid strain was sporulated and haploid colonies were then selected via replica plating onto selective media to select relevant haploid mutant strains. These strains were also used in our attempts by PCR to generate deletion cassettes. BY4741a strains were used to generate VPs75 and MAK31 deletion mutants by homologous recombination with deletion cassettes that we created using primers containing 5' ends having homology to the gene region and amplifying either a TRP1 or LEU2 cassette from plasmids.

Daily Cell Density (cells/mL) For EST2, VPS75 and MAK31 Knockouts

	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	Day 7	Day 8	Day 9	Day 10
1296	61000000	21400000	85066666	58666666	116866666	574666667	78133333	106866666	83066666	86500000
VPS75	134500000	120000000	113833333	63800000	72433333	111500000	113700000	124100000	115000000	115400000
MAK31	131800000	118333333	117833333	145166666	118733333	120066666	117733333	125333333	116400000	120633333

The results for VPS75 and MAK31 show our predicted results. After the growth curve decreased on day 3 for VPS75, it increased again on day 4. The VPS75 growth curve leveled off after day 6. The MAK31 growth curve fluctuated a bit, but showed a general trend of decrease and then increase.

Daily Cell Density (cells/mL) For EST2 and EST2/RAD52 Knockouts

	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
W303A	137600000	114500000	134050000	129750000	109050000	118450000
ΔEST2 (S21)	115900000	16100000	13800000	55866666.67	118600000	102766666.7
ΔEST2 (S22)	112300000	33433333.33	15133333.33	95033333.33	48900000	109500000
ΔRAD52/EST2	107500000	13266666.67	12366666.67	10933333.33	20433333.33	11233333.33