Team:Buenos Aires/Results/Strains

In the table we can see 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)

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.

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 cel or 102 cel 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.

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 Table, we have a very low, but existent, number of revertants from both his and trp auxotrophy strains. This number should be taken into account when seeing results from coculture growth after several days, given that the rate of revertants in liquid medium may as well be of the same numerical order. Growth in coculture was high and near to the number we expected as result of natural cooperation, which confirms that there is a natural way in which the strains cooperate by sharing each other´s missing aminoacids. This is encouraging since we know confirmed that each strain is able to see and relate with each other and therefore we have an open communication way over which to work and control with engineering.

Measurement of Trp in medium and Basal Production

To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan excreted by natural strains to the medium, so we can compare. With that end in mind, we designed a protocol for measurement of tryptophan in medium, based in its fluorescense at 350nm, when excited with 295nm light. As a previous step, we checked that none of the other aminoacids used in the medium interferes, by graphically comparing the spectres for uncomplemented medium and medium complemented with leucine, uracile and histidine, at an appropiate range.

To determine Trp concentration, we must first have a way to transform our readings (intensity) to a more useful output, so we made a calibration curve, through serialized 1:2 dilutions of our medium, which Trp's concentration is 50mg/mL, until approximately constant intensity.

The procedure to measure secretion rates will be growing the strain from a known OD in exponential growth phase in -T medium and plotting it's OD over time, spin-drying at time=t, retrieving the supernatant's Trp concentration and dividing it by the integral of OD vs. time between time=0 and time=t, so we get to a rate which will be proportional to the number of cells in the culture, which means we can actually compare between different strains. Since our medium is free from Trp, all of it should come from within the cells, and if the culture is growing at exponential rates, lysis should be negligible, so the only explanation would be cells exporting their own Trp.

Graph:Tryptophan calibration curve

Results

As can be seen from the graph the screening of the concentration of the Trp in medium describes an almost lineal function. Through this experiment we can be sure that we would be able to measure increase of Trp in medium as it is exported from the cells, within the biological range of export. The sensitivity of this method seems to be enough to detect concentrations as low as ~0.02mg/mL, and as high as 50mg/mL, maybe more. Since our medium is 50mg/mL, we assume that's the saturation point of the curve. If we get bigger intensities than the one corresponding to it, we will dilute the sample.

Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.

Growth dependence on the Trp and His concentrations

A important thing to characterize of the system is the dependence of the growth rate of the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His). To do this we measured the final OD after an overnight growth in medium with different concentrations of Trp and His.

We used strain ACL-379, that is auxotroph for both Trp and His. We prepared serial dilutions of SC medium in –T and –H medium, therefore creating two curves: one with decreasing concentrations of Trp and the other with decreasing concentrations of His. We then inoculated equal amounts of ACL-379 in each tube and incubated them overnight at 30°C with agitation. We took a picture of each tube and measured the OD600 reached by each culture.

Results

As expected the growth has a sigmoidal relationship with the concentration of Trp and His, when plotted in semilogarithmic scale. We call EC50 the effective concentration of each aminoacid at which the culture reaches 50% of the maximal growth. We considered these values as proxies of the Khis and Ktrp parameters of the mathematical model, which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding.

These results can also be observed by comparison of images that show the tubes at different OD.

Images from HLC series	Images from TLC series

Notes: SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.

HTLC is the culture in the medium with all the required aminoacids.

S(Number) are the serial dilutions of HTLC with medium that lacks Histidine (HLC) and Tryptophane (TLC.

HLC and TLC are mediums without cells.