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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

SynEco Testing

Coculture of transformed strains

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

We designed an assay to do so; following the growth of these strains:

CFP_His	YFP_TRPb

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

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.