Team:Buenos Aires/Results/Strains


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Strain characterization

a) Description of strains

Through our experiments we worked with the following strains kindly provided by Alejandro Colman-Lerner's Lab:

Strain ID Relevant Auxotrophies Fluorescence Used as
TCY 3043 (-H-T) No fluorescence Negative control
TCY 3190 (+H-T) YFP + (Induced CFP) For coculture
TCY 3265 (-H+T) CFP For coculture
TCY 3154 (+H+T) CFP +(induced YFP) Positive Control

Table: Hystidine (H) and Tryptophane (T) auxotrophies per strain, type of fluorescence and description of most common utilization during the experiments. 

Nearly 15 other similar strains were evaluated and discarded due to several reasons (low screening potentiality; requirement of hormones for fluorescence induction; high reverting rate of auxotrophies, among others)

Foto 1

b) Fluorescence screening

We measured Strains 3281 (YFP) and 3265 (CFP) and got a spectrum of each one prooving that these strains can be distinguished by their fluorescence in culture.

Graficos 1

c) Auxotrophy confirmation

Several times during the experiments we control and checked if the auxotrophies in the selected strain were functional by plating all of them in medium deficient in aminoacids (-H; -T; -H-T and control +H+T). We observed differential growth according to expected due to the description of each strain in point a)

Foto 2,3,4,5

Figures a-d: Auxotrophies check. Plate a : medium complete. Plate b: medium without T. Plate c: medium without H and T. Plate d: medium without H.

We observed all the strains grew in Plate a and only 3154 (+H+T) grew in Plate c. In plate B, only those strains able to synthesize T grew (3265 and 3154) and in Plate d, only those able to produce H grew (3190 and 3154), as expected. This means our strains work according to their description. We did this several times during the months to check for reversions or contaminations.

d) Coculture in liquid medium: growth

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

i) 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-):(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

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

Graficos 2,3,4

As shown in graphs 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.

ii) 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.

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

Table: Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2).

Grafico 5

Picture Wednedsday

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.