Team:Buenos Aires/Results/SynEcoTesting
From 2012.igem.org
Contents 
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 

Protocol


384 wells plate to be used for epifluorescence microscope. 
Results
Coculture in liquid medium
We used for these experiment TCY3190(H+T) and TCY3265(HT+) Positive control: TCY3154 (H+T+) and negative control TCY3043(HT)
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 Proportion (H+T):(HT+)  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.
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 crossfeeding 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 +HT  Control of Trp revertants  2  7 
()  ()  Strain +HT  Strain –H+T  Coculture; we expect to see natural cooperation  960  800 
()  ()  Strain –H+T  Strain +HT  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 –HT 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.