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Contents 1 SynEco Testing 1.1 Coculture of transformed strains 1.1.1 Protocol 1.1.2 Results 1.2 Coculture in liquid medium 1.2.1 At different initial OD and proportions 1.2.2 At the same initial OD: 0.2, followed over time 1.3 Coculture in Agar and Revertant mutation control 1.3.1 Results

SynEco Testing

Coculture of transformed strains

We did the first assay to test whether our system works and how two transformed strains grow together.

Transformed cells coculture and controls. Treatment Strain A Strain B 1 YFP_TRPb_TRPZipper2 CFP_HIS_PolyHa 2 YFP_TRPb_TRPZipper2 CFP_HIS 3 YFP_TRPb_TRPZipper2 4 YFP_TRPb CFP_HIS_PolyHa 5 CFP_HIS_PolyHa 6 YFP_TRPb CFP_HIS 7 CFP_HIS 8 YFP_TRPb

Protocol

Starters of strains were grown over night at 30°C, according the scheme showed in the table The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase. Cells were centrifugated and then washed with medium –HT. We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl. Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells. We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.

Strain Media YFP_TRPb_TRPZipper2 -T YFP_TRPb -T CFP_HIS_PolyHa -H CFP_HIS -H

384 wells plate to be used for epifluorescence microscope.

Results

In the time framed we worked with, we werent able to see significant differences in the cell density. We are aware, based on our model predictions that there could be a lag time of days (1-3). This is also consistent with the results obtained in other experiments. We will repeat this test in a longer time period.

Coculture in liquid medium

We used for these experiment TCY3190(H+T-) and TCY3265(H-T+) Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)

At different initial OD and proportions

Cultures were set at different initial concentrations (0.25, 0.1 and 0.01) and proportions (1:1; 1:9; 9:1). After 24 hs, we measured OD with the use of a spectrophotometer (two replicas) and we calculated the mean OD and a Growth factor (as Mean OD en time 1 over Initial OD time 0).

Coculture at different initial OD and proportions (Days 0 and 1)

Coculture Proportion (H+T-):(H-T+)	Initial OD(t=0)	OD1 (t=1)	OD2 (t=1)	dilution used for measure t=1	Mean OD	Growth Factor
01:01	0,25	0,32	0,314	10	3,17	12,68
09:01	0,25	0,148	0,144	10	1,46	5,84
01:09	0,25	0,138	0,189	10	1,635	6,54
01:01	0,1	0,109	0,169	10	1,39	13,9
09:01	0,1	0,04	0,045	10	0,425	4,25
01:09	0,1	0,067	0,053	10	0,6	6
01:01	0,01	0,067	0,061	1	0,064	6,4
09:01	0,01	0,056	0,05	1	0,053	5,3
01:09	0,01	0,074	0,073	1	0,0735	7,35

As shown in graph and table there is a basal growth that does not depend on the initial OD or strain proportion, of a growth factor of 6 approximately. However we observed a much higher growth at the proportion 1:1 when the initial OD 0.25 and 0.1. Therefore we can assume that at these proportions there is a natural cooperation between the strains and that should be the level of growth that we would like to assess through our bioengineering. Besides we would like to be able in the future to tune the strains in order to be able to obtain in the proportions 9:1 and 1:9 similar results to those obtained in the 1:1, at our own will.

At the same initial OD: 0.2, followed over time

We set the same cultures and cocultures as in point i), but starting all of them at the same OD: 0.2 and we followed them over 2 days. At day 1 we took pictures of them and at day 2 we measured the final OD.

Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2) Strain Day 0 Day 2 TCY 3190 (-H+T) 0,2 2,92 TCY 3265 (+H-T) 0,2 0,19 Coculture of strains (TCY 3190- TCY 3265) 0,2 2,76 Negative control (TCY 3043 / -H-T) 0,2 0,6 Positive Control (TCY 3154/ +H+T) 0,2 2,54 Picture: Day 1 after starting cultures, shows different OD reached by strains. We repeated this experiment 4 times with different modifications: increasing the amount of days for up to a week, measuring every 12 hs instead of every 24 hs and using different strains. However, bacterial contaminations and the high rate of revertants prevented us from getting to a valid results in those cases, whereas the experiment up to day 2 always worked correctly. This denotes that we should assess the problem of contamination (for example including ampicilin in the cultures) and revertant rate (revising the design of the experiment or looking for more stable strains) as the impossibility to go further than day 2 may put limitations to some applications of the Synthetic Community.

Coculture in Agar and Revertant mutation control

Through this experiment we aim to quantify the rate of revertants of each strain, and to asses if cross-feeding between a lawn of cells of one strain and colonies from and other strain is posible.

We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.

We started a culture of each strain in synthetic complete medium, measured its OD 24 hs after the culture initiated, replaced the synthetic complete medium for one lacking both H and T (to avoid residual growth) and plated ~10^6 cells (lawn) or ~10^2 cells (seed) as shown by the following table (we considered OD600=1 represents 3*10^7 cells). At the same time, 3 controls (one for each strain) were carried in YPD complete medium to check the viability of each strain separately, and to estimate the seed CFU (colony formin units) more precisely.

Medium H	Medium T	Lawn (10^6 cells)	Seed (10^2 cells)	Description of experiment	Results after 3 days - Replica 1	Results after 3 days - Replica 2
(-)	(+)	(-)	Strain –H+T	Control of His revertants	7	7
(+)	(-)	(-)	Strain +H-T	Control of Trp revertants	2	7
(-)	(-)	Strain +H-T	Strain –H+T	Coculture; we expect to see natural cooperation	960	800
(-)	(-)	Strain –H+T	Strain +H-T	Coculture; we expect to see natural cooperation	500	712
(-)	(-)	(-)	Strain +H+T	Viability of yeasts in medium	171	(-)

Table: Shows description of each plate content and results in number of colonies counted by plate at day 3. YPD control results plates are not shown in the table.

Petri Dishes	With marks of the counting of colonies

Results

The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. As shown in the table, we have a low, but existent, number of revertants from both his and trp auxotrophy strains. This number should be taken into account when interpreting the results from coculture growth after several days, given that the rate of revertants in liquid medium is probably the same.

Growth in coculture was puzzling, as it resulted in more colonies than the expected. If cooperation was effective, we expected to see as many colonies as "seed" cells, not more. Revertion of cells from the "lawn" doesn't explain the number of colonies either. Probably a combination of both these effects are taking place.