http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Abush842012.igem.org - User contributions [en]2024-03-29T06:46:53ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-10-27T04:04:47Z<p>Abush84: /* At different initial OD and proportions */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|-<br />
|TCY 3128<br />
| style="text-align: center;" |(-H-T)<br />
|CFP <br />
|His device testing<br />
|-<br />
|TCY 3081<br />
| style="text-align: center;" |(-H-T)<br />
|YFP <br />
|Trp device testing<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurement of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|300px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Fluoro- igembsas2012. strains.png|650px]]<br />
|-<br />
|'''CFP Fluorescence Screening and YFP Fluorescence Screening'''<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase. Therefore we decided to use epifluorescence microscopy (see below).<br />
<br />
== Strains proportion measurement ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains TCY-3281 (that expresses YFP) and TCY-3265 (that expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control Mix 2: 80% CFP; 20%YFP Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP Mix 5: 40% CFP; 60%YFP Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|100px]]<br />
|[[File:Bsas2012-strains-figura2.png|100px]]<br />
|[[File:Bsas2012-strains-figura4.png|100px]]<br />
|[[File:Bsas2012-strains-figura5.png|100px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:HIS-BSAS2012.png|400px]]<br />
|}<br />
<br />
<br />
As shown in the figure and table there is a basal growth that does not depend on the initial OD or strain proportion. This residual growth produces a growth factor of 6 approximately.<br />
However we observed a much higher growth at the proportion 1:1 when the initial OD 0.25 and 0.1. This suggests that at these proportions there is a natural cooperation between the strains. The objective of the project is to build upon this natural cooperation and to allow for tunable proportions.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
|}<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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 20μg/ml, until approximately constant intensity.<br />
<br />
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 and diffusing their own Trp.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Curva.png | 250px]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
The sensitivity of this method seems to be enough to detect concentrations as low as ~0.01μg/ml, and as high as 20μg/ml, maybe more. Since our medium is 20μg/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.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC (no cells)<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
| -T<br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
| Trp/2<br />
|2.56 <br />
|2.17<br />
|-<br />
| Trp/4<br />
|3.01 <br />
|3.11<br />
|-<br />
|Trp/8<br />
|1.54 <br />
|1.55<br />
|-<br />
|Trp/16<br />
|0.393 <br />
|0.409<br />
|-<br />
|Trp/32<br />
|0.013 <br />
|0.003<br />
|-<br />
| -H<br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
| His/2 <br />
|3.68 <br />
|3.84<br />
|-<br />
| His/4<br />
|2.07 <br />
|2.00<br />
|-<br />
|His/8<br />
|1.17 <br />
|0.97<br />
|-<br />
|His/16 <br />
|0.47 <br />
|0.432<br />
|-<br />
|His/32 <br />
|0.238 <br />
|0.257<br />
|-<br />
|SC (w/cells) <br />
|4.88 <br />
|4.91<br />
|} <br />
<br />
==== Results ====<br />
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 [[Team:Buenos_Aires/Project/Model#Parameter_selection|mathematical model]], which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|250px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|250px]] <br />
|-<br />
|Images from HLU series<br />
|Images from TLU series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLU is the culture in the medium with all the required aminoacids.<br />
<br />
<br />
== Experimental determination of strains death rate==<br />
<br />
We set out to determine how long can auxotroph cells[link] survive in media that lacks both Trytophan and Histidine. These values are the '''death''' parameters for CFP and YFP strains used in our model[link]. These were taken as equal in the mathematical analysis for simplicity but now we would like to test whether this approximation is accurate.<br />
<br />
Given that our system most likely will present a lag phase until a certain amount of both AmioAcids is accumulated in the media, will the cells be viable until this occurs? This is a neccesary check of our '' system's feasability''.<br />
<br />
===== Protocol =====<br />
<br />
For this experiment we used<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-YFP.jpg|200px]]<br />
|[[File:BsAs2012-icono-CFP.jpg|200px]]<br />
|- align="center"<br />
|YFP Strain<br />
|CFP Strain<br />
|}<br />
<br />
*We set cultures of the two auxotroph strains without being transformed (YFP and CFP) in medium –HT at an initial OD of 0.01. <br />
*Each day we plated the same amount of µl of the culture and counted the number of colonies obtain in each plate. We set 3 replica of each strain.<br />
<br />
===== Result =====<br />
<br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" |Strain<br />
! scope="row" style="background: #7ac5e8" |Replica<br />
! scope="row" style="background: #7ac5e8" |Monday<br />
! scope="row" style="background: #7ac5e8" |Tuesday<br />
! scope="row" style="background: #7ac5e8" |Wednesday<br />
|-<br />
|CFP<br />
|1<br />
|260<br />
|320<br />
|285<br />
|-<br />
|CFP<br />
|2<br />
|267<br />
|314<br />
|76<br />
|-<br />
|CFP<br />
|3<br />
|413<br />
|362<br />
|278<br />
|-<br />
|YFP<br />
|1<br />
|230<br />
|316<br />
|688<br />
|-<br />
|YFP<br />
|2<br />
|291<br />
|194<br />
|524<br />
|-<br />
|YFP<br />
|3<br />
|449<br />
|344<br />
|725<br />
|}<br />
<br />
'''Table:''' Number of colonies counted per plate.<br />
<br />
We expected to see a decrease in the number of colonies because of cell death. We found that this was not the case in the experiment's time lapse. However we observed that the size of the colonies was smaller everyday as can be seen in the following pictures.<br />
<br />
[[File:Bsas2012kdeathcells.png| 500px]]<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a persistant state. In this way cells can survive for a period of time in media defficient in amino acid (at least, during the time course of our experiment), but grow slower. Probably this would require more time than 3 days to observe significative cell dying.<br />
<br />
These results are consistent with the chosen parameters. Moreover, the slower the death rate the bigger the area in the Parameter Space where regulation is feasable.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-10-27T03:59:21Z<p>Abush84: /* Strain proportion measurement */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|-<br />
|TCY 3128<br />
| style="text-align: center;" |(-H-T)<br />
|CFP <br />
|His device testing<br />
|-<br />
|TCY 3081<br />
| style="text-align: center;" |(-H-T)<br />
|YFP <br />
|Trp device testing<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurement of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|300px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Fluoro- igembsas2012. strains.png|650px]]<br />
|-<br />
|'''CFP Fluorescence Screening and YFP Fluorescence Screening'''<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase. Therefore we decided to use epifluorescence microscopy (see below).<br />
<br />
== Strains proportion measurement ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains TCY-3281 (that expresses YFP) and TCY-3265 (that expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control Mix 2: 80% CFP; 20%YFP Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP Mix 5: 40% CFP; 60%YFP Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|100px]]<br />
|[[File:Bsas2012-strains-figura2.png|100px]]<br />
|[[File:Bsas2012-strains-figura4.png|100px]]<br />
|[[File:Bsas2012-strains-figura5.png|100px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:HIS-BSAS2012.png|400px]]<br />
|}<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
|}<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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 20μg/ml, until approximately constant intensity.<br />
<br />
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 and diffusing their own Trp.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Curva.png | 250px]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
The sensitivity of this method seems to be enough to detect concentrations as low as ~0.01μg/ml, and as high as 20μg/ml, maybe more. Since our medium is 20μg/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.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC (no cells)<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
| -T<br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
| Trp/2<br />
|2.56 <br />
|2.17<br />
|-<br />
| Trp/4<br />
|3.01 <br />
|3.11<br />
|-<br />
|Trp/8<br />
|1.54 <br />
|1.55<br />
|-<br />
|Trp/16<br />
|0.393 <br />
|0.409<br />
|-<br />
|Trp/32<br />
|0.013 <br />
|0.003<br />
|-<br />
| -H<br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
| His/2 <br />
|3.68 <br />
|3.84<br />
|-<br />
| His/4<br />
|2.07 <br />
|2.00<br />
|-<br />
|His/8<br />
|1.17 <br />
|0.97<br />
|-<br />
|His/16 <br />
|0.47 <br />
|0.432<br />
|-<br />
|His/32 <br />
|0.238 <br />
|0.257<br />
|-<br />
|SC (w/cells) <br />
|4.88 <br />
|4.91<br />
|} <br />
<br />
==== Results ====<br />
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 [[Team:Buenos_Aires/Project/Model#Parameter_selection|mathematical model]], which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|250px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|250px]] <br />
|-<br />
|Images from HLU series<br />
|Images from TLU series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLU is the culture in the medium with all the required aminoacids.<br />
<br />
<br />
== Experimental determination of strains death rate==<br />
<br />
We set out to determine how long can auxotroph cells[link] survive in media that lacks both Trytophan and Histidine. These values are the '''death''' parameters for CFP and YFP strains used in our model[link]. These were taken as equal in the mathematical analysis for simplicity but now we would like to test whether this approximation is accurate.<br />
<br />
Given that our system most likely will present a lag phase until a certain amount of both AmioAcids is accumulated in the media, will the cells be viable until this occurs? This is a neccesary check of our '' system's feasability''.<br />
<br />
===== Protocol =====<br />
<br />
For this experiment we used<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-YFP.jpg|200px]]<br />
|[[File:BsAs2012-icono-CFP.jpg|200px]]<br />
|- align="center"<br />
|YFP Strain<br />
|CFP Strain<br />
|}<br />
<br />
*We set cultures of the two auxotroph strains without being transformed (YFP and CFP) in medium –HT at an initial OD of 0.01. <br />
*Each day we plated the same amount of µl of the culture and counted the number of colonies obtain in each plate. We set 3 replica of each strain.<br />
<br />
===== Result =====<br />
<br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" |Strain<br />
! scope="row" style="background: #7ac5e8" |Replica<br />
! scope="row" style="background: #7ac5e8" |Monday<br />
! scope="row" style="background: #7ac5e8" |Tuesday<br />
! scope="row" style="background: #7ac5e8" |Wednesday<br />
|-<br />
|CFP<br />
|1<br />
|260<br />
|320<br />
|285<br />
|-<br />
|CFP<br />
|2<br />
|267<br />
|314<br />
|76<br />
|-<br />
|CFP<br />
|3<br />
|413<br />
|362<br />
|278<br />
|-<br />
|YFP<br />
|1<br />
|230<br />
|316<br />
|688<br />
|-<br />
|YFP<br />
|2<br />
|291<br />
|194<br />
|524<br />
|-<br />
|YFP<br />
|3<br />
|449<br />
|344<br />
|725<br />
|}<br />
<br />
'''Table:''' Number of colonies counted per plate.<br />
<br />
We expected to see a decrease in the number of colonies because of cell death. We found that this was not the case in the experiment's time lapse. However we observed that the size of the colonies was smaller everyday as can be seen in the following pictures.<br />
<br />
[[File:Bsas2012kdeathcells.png| 500px]]<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a persistant state. In this way cells can survive for a period of time in media defficient in amino acid (at least, during the time course of our experiment), but grow slower. Probably this would require more time than 3 days to observe significative cell dying.<br />
<br />
These results are consistent with the chosen parameters. Moreover, the slower the death rate the bigger the area in the Parameter Space where regulation is feasable.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-10-27T03:58:42Z<p>Abush84: /* Screening of strain proportion */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|-<br />
|TCY 3128<br />
| style="text-align: center;" |(-H-T)<br />
|CFP <br />
|His device testing<br />
|-<br />
|TCY 3081<br />
| style="text-align: center;" |(-H-T)<br />
|YFP <br />
|Trp device testing<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurement of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|300px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Fluoro- igembsas2012. strains.png|650px]]<br />
|-<br />
|'''CFP Fluorescence Screening and YFP Fluorescence Screening'''<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase. Therefore we decided to use epifluorescence microscopy (see below).<br />
<br />
== Strain proportion measurement ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains TCY-3281 (that expresses YFP) and TCY-3265 (that expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control Mix 2: 80% CFP; 20%YFP Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP Mix 5: 40% CFP; 60%YFP Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|100px]]<br />
|[[File:Bsas2012-strains-figura2.png|100px]]<br />
|[[File:Bsas2012-strains-figura4.png|100px]]<br />
|[[File:Bsas2012-strains-figura5.png|100px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:HIS-BSAS2012.png|400px]]<br />
|}<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
|}<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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 20μg/ml, until approximately constant intensity.<br />
<br />
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 and diffusing their own Trp.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Curva.png | 250px]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
The sensitivity of this method seems to be enough to detect concentrations as low as ~0.01μg/ml, and as high as 20μg/ml, maybe more. Since our medium is 20μg/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.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC (no cells)<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
| -T<br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
| Trp/2<br />
|2.56 <br />
|2.17<br />
|-<br />
| Trp/4<br />
|3.01 <br />
|3.11<br />
|-<br />
|Trp/8<br />
|1.54 <br />
|1.55<br />
|-<br />
|Trp/16<br />
|0.393 <br />
|0.409<br />
|-<br />
|Trp/32<br />
|0.013 <br />
|0.003<br />
|-<br />
| -H<br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
| His/2 <br />
|3.68 <br />
|3.84<br />
|-<br />
| His/4<br />
|2.07 <br />
|2.00<br />
|-<br />
|His/8<br />
|1.17 <br />
|0.97<br />
|-<br />
|His/16 <br />
|0.47 <br />
|0.432<br />
|-<br />
|His/32 <br />
|0.238 <br />
|0.257<br />
|-<br />
|SC (w/cells) <br />
|4.88 <br />
|4.91<br />
|} <br />
<br />
==== Results ====<br />
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 [[Team:Buenos_Aires/Project/Model#Parameter_selection|mathematical model]], which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|250px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|250px]] <br />
|-<br />
|Images from HLU series<br />
|Images from TLU series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLU is the culture in the medium with all the required aminoacids.<br />
<br />
<br />
== Experimental determination of strains death rate==<br />
<br />
We set out to determine how long can auxotroph cells[link] survive in media that lacks both Trytophan and Histidine. These values are the '''death''' parameters for CFP and YFP strains used in our model[link]. These were taken as equal in the mathematical analysis for simplicity but now we would like to test whether this approximation is accurate.<br />
<br />
Given that our system most likely will present a lag phase until a certain amount of both AmioAcids is accumulated in the media, will the cells be viable until this occurs? This is a neccesary check of our '' system's feasability''.<br />
<br />
===== Protocol =====<br />
<br />
For this experiment we used<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-YFP.jpg|200px]]<br />
|[[File:BsAs2012-icono-CFP.jpg|200px]]<br />
|- align="center"<br />
|YFP Strain<br />
|CFP Strain<br />
|}<br />
<br />
*We set cultures of the two auxotroph strains without being transformed (YFP and CFP) in medium –HT at an initial OD of 0.01. <br />
*Each day we plated the same amount of µl of the culture and counted the number of colonies obtain in each plate. We set 3 replica of each strain.<br />
<br />
===== Result =====<br />
<br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" |Strain<br />
! scope="row" style="background: #7ac5e8" |Replica<br />
! scope="row" style="background: #7ac5e8" |Monday<br />
! scope="row" style="background: #7ac5e8" |Tuesday<br />
! scope="row" style="background: #7ac5e8" |Wednesday<br />
|-<br />
|CFP<br />
|1<br />
|260<br />
|320<br />
|285<br />
|-<br />
|CFP<br />
|2<br />
|267<br />
|314<br />
|76<br />
|-<br />
|CFP<br />
|3<br />
|413<br />
|362<br />
|278<br />
|-<br />
|YFP<br />
|1<br />
|230<br />
|316<br />
|688<br />
|-<br />
|YFP<br />
|2<br />
|291<br />
|194<br />
|524<br />
|-<br />
|YFP<br />
|3<br />
|449<br />
|344<br />
|725<br />
|}<br />
<br />
'''Table:''' Number of colonies counted per plate.<br />
<br />
We expected to see a decrease in the number of colonies because of cell death. We found that this was not the case in the experiment's time lapse. However we observed that the size of the colonies was smaller everyday as can be seen in the following pictures.<br />
<br />
[[File:Bsas2012kdeathcells.png| 500px]]<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a persistant state. In this way cells can survive for a period of time in media defficient in amino acid (at least, during the time course of our experiment), but grow slower. Probably this would require more time than 3 days to observe significative cell dying.<br />
<br />
These results are consistent with the chosen parameters. Moreover, the slower the death rate the bigger the area in the Parameter Space where regulation is feasable.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-10-27T03:57:00Z<p>Abush84: /* Measurement of strains fluorescence */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|-<br />
|TCY 3128<br />
| style="text-align: center;" |(-H-T)<br />
|CFP <br />
|His device testing<br />
|-<br />
|TCY 3081<br />
| style="text-align: center;" |(-H-T)<br />
|YFP <br />
|Trp device testing<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurement of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|300px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Fluoro- igembsas2012. strains.png|650px]]<br />
|-<br />
|'''CFP Fluorescence Screening and YFP Fluorescence Screening'''<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase. Therefore we decided to use epifluorescence microscopy (see below).<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control Mix 2: 80% CFP; 20%YFP Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP Mix 5: 40% CFP; 60%YFP Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|100px]]<br />
|[[File:Bsas2012-strains-figura2.png|100px]]<br />
|[[File:Bsas2012-strains-figura4.png|100px]]<br />
|[[File:Bsas2012-strains-figura5.png|100px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:HIS-BSAS2012.png|400px]]<br />
|}<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
|}<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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 20μg/ml, until approximately constant intensity.<br />
<br />
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 and diffusing their own Trp.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Curva.png | 250px]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
The sensitivity of this method seems to be enough to detect concentrations as low as ~0.01μg/ml, and as high as 20μg/ml, maybe more. Since our medium is 20μg/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.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC (no cells)<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
| -T<br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
| Trp/2<br />
|2.56 <br />
|2.17<br />
|-<br />
| Trp/4<br />
|3.01 <br />
|3.11<br />
|-<br />
|Trp/8<br />
|1.54 <br />
|1.55<br />
|-<br />
|Trp/16<br />
|0.393 <br />
|0.409<br />
|-<br />
|Trp/32<br />
|0.013 <br />
|0.003<br />
|-<br />
| -H<br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
| His/2 <br />
|3.68 <br />
|3.84<br />
|-<br />
| His/4<br />
|2.07 <br />
|2.00<br />
|-<br />
|His/8<br />
|1.17 <br />
|0.97<br />
|-<br />
|His/16 <br />
|0.47 <br />
|0.432<br />
|-<br />
|His/32 <br />
|0.238 <br />
|0.257<br />
|-<br />
|SC (w/cells) <br />
|4.88 <br />
|4.91<br />
|} <br />
<br />
==== Results ====<br />
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 [[Team:Buenos_Aires/Project/Model#Parameter_selection|mathematical model]], which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|250px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|250px]] <br />
|-<br />
|Images from HLU series<br />
|Images from TLU series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLU is the culture in the medium with all the required aminoacids.<br />
<br />
<br />
== Experimental determination of strains death rate==<br />
<br />
We set out to determine how long can auxotroph cells[link] survive in media that lacks both Trytophan and Histidine. These values are the '''death''' parameters for CFP and YFP strains used in our model[link]. These were taken as equal in the mathematical analysis for simplicity but now we would like to test whether this approximation is accurate.<br />
<br />
Given that our system most likely will present a lag phase until a certain amount of both AmioAcids is accumulated in the media, will the cells be viable until this occurs? This is a neccesary check of our '' system's feasability''.<br />
<br />
===== Protocol =====<br />
<br />
For this experiment we used<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-YFP.jpg|200px]]<br />
|[[File:BsAs2012-icono-CFP.jpg|200px]]<br />
|- align="center"<br />
|YFP Strain<br />
|CFP Strain<br />
|}<br />
<br />
*We set cultures of the two auxotroph strains without being transformed (YFP and CFP) in medium –HT at an initial OD of 0.01. <br />
*Each day we plated the same amount of µl of the culture and counted the number of colonies obtain in each plate. We set 3 replica of each strain.<br />
<br />
===== Result =====<br />
<br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" |Strain<br />
! scope="row" style="background: #7ac5e8" |Replica<br />
! scope="row" style="background: #7ac5e8" |Monday<br />
! scope="row" style="background: #7ac5e8" |Tuesday<br />
! scope="row" style="background: #7ac5e8" |Wednesday<br />
|-<br />
|CFP<br />
|1<br />
|260<br />
|320<br />
|285<br />
|-<br />
|CFP<br />
|2<br />
|267<br />
|314<br />
|76<br />
|-<br />
|CFP<br />
|3<br />
|413<br />
|362<br />
|278<br />
|-<br />
|YFP<br />
|1<br />
|230<br />
|316<br />
|688<br />
|-<br />
|YFP<br />
|2<br />
|291<br />
|194<br />
|524<br />
|-<br />
|YFP<br />
|3<br />
|449<br />
|344<br />
|725<br />
|}<br />
<br />
'''Table:''' Number of colonies counted per plate.<br />
<br />
We expected to see a decrease in the number of colonies because of cell death. We found that this was not the case in the experiment's time lapse. However we observed that the size of the colonies was smaller everyday as can be seen in the following pictures.<br />
<br />
[[File:Bsas2012kdeathcells.png| 500px]]<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a persistant state. In this way cells can survive for a period of time in media defficient in amino acid (at least, during the time course of our experiment), but grow slower. Probably this would require more time than 3 days to observe significative cell dying.<br />
<br />
These results are consistent with the chosen parameters. Moreover, the slower the death rate the bigger the area in the Parameter Space where regulation is feasable.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-10-27T03:54:53Z<p>Abush84: /* Description of strains */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|-<br />
|TCY 3128<br />
| style="text-align: center;" |(-H-T)<br />
|CFP <br />
|His device testing<br />
|-<br />
|TCY 3081<br />
| style="text-align: center;" |(-H-T)<br />
|YFP <br />
|Trp device testing<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurement of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|300px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Fluoro- igembsas2012. strains.png|650px]]<br />
|-<br />
|'''CFP Fluorescence Screening and YFP Fluorescence Screening'''<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control Mix 2: 80% CFP; 20%YFP Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP Mix 5: 40% CFP; 60%YFP Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|100px]]<br />
|[[File:Bsas2012-strains-figura2.png|100px]]<br />
|[[File:Bsas2012-strains-figura4.png|100px]]<br />
|[[File:Bsas2012-strains-figura5.png|100px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:HIS-BSAS2012.png|400px]]<br />
|}<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
|}<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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 20μg/ml, until approximately constant intensity.<br />
<br />
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 and diffusing their own Trp.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Curva.png | 250px]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
The sensitivity of this method seems to be enough to detect concentrations as low as ~0.01μg/ml, and as high as 20μg/ml, maybe more. Since our medium is 20μg/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.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC (no cells)<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
| -T<br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
| Trp/2<br />
|2.56 <br />
|2.17<br />
|-<br />
| Trp/4<br />
|3.01 <br />
|3.11<br />
|-<br />
|Trp/8<br />
|1.54 <br />
|1.55<br />
|-<br />
|Trp/16<br />
|0.393 <br />
|0.409<br />
|-<br />
|Trp/32<br />
|0.013 <br />
|0.003<br />
|-<br />
| -H<br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
| His/2 <br />
|3.68 <br />
|3.84<br />
|-<br />
| His/4<br />
|2.07 <br />
|2.00<br />
|-<br />
|His/8<br />
|1.17 <br />
|0.97<br />
|-<br />
|His/16 <br />
|0.47 <br />
|0.432<br />
|-<br />
|His/32 <br />
|0.238 <br />
|0.257<br />
|-<br />
|SC (w/cells) <br />
|4.88 <br />
|4.91<br />
|} <br />
<br />
==== Results ====<br />
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 [[Team:Buenos_Aires/Project/Model#Parameter_selection|mathematical model]], which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|250px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|250px]] <br />
|-<br />
|Images from HLU series<br />
|Images from TLU series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLU is the culture in the medium with all the required aminoacids.<br />
<br />
<br />
== Experimental determination of strains death rate==<br />
<br />
We set out to determine how long can auxotroph cells[link] survive in media that lacks both Trytophan and Histidine. These values are the '''death''' parameters for CFP and YFP strains used in our model[link]. These were taken as equal in the mathematical analysis for simplicity but now we would like to test whether this approximation is accurate.<br />
<br />
Given that our system most likely will present a lag phase until a certain amount of both AmioAcids is accumulated in the media, will the cells be viable until this occurs? This is a neccesary check of our '' system's feasability''.<br />
<br />
===== Protocol =====<br />
<br />
For this experiment we used<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-YFP.jpg|200px]]<br />
|[[File:BsAs2012-icono-CFP.jpg|200px]]<br />
|- align="center"<br />
|YFP Strain<br />
|CFP Strain<br />
|}<br />
<br />
*We set cultures of the two auxotroph strains without being transformed (YFP and CFP) in medium –HT at an initial OD of 0.01. <br />
*Each day we plated the same amount of µl of the culture and counted the number of colonies obtain in each plate. We set 3 replica of each strain.<br />
<br />
===== Result =====<br />
<br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" |Strain<br />
! scope="row" style="background: #7ac5e8" |Replica<br />
! scope="row" style="background: #7ac5e8" |Monday<br />
! scope="row" style="background: #7ac5e8" |Tuesday<br />
! scope="row" style="background: #7ac5e8" |Wednesday<br />
|-<br />
|CFP<br />
|1<br />
|260<br />
|320<br />
|285<br />
|-<br />
|CFP<br />
|2<br />
|267<br />
|314<br />
|76<br />
|-<br />
|CFP<br />
|3<br />
|413<br />
|362<br />
|278<br />
|-<br />
|YFP<br />
|1<br />
|230<br />
|316<br />
|688<br />
|-<br />
|YFP<br />
|2<br />
|291<br />
|194<br />
|524<br />
|-<br />
|YFP<br />
|3<br />
|449<br />
|344<br />
|725<br />
|}<br />
<br />
'''Table:''' Number of colonies counted per plate.<br />
<br />
We expected to see a decrease in the number of colonies because of cell death. We found that this was not the case in the experiment's time lapse. However we observed that the size of the colonies was smaller everyday as can be seen in the following pictures.<br />
<br />
[[File:Bsas2012kdeathcells.png| 500px]]<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a persistant state. In this way cells can survive for a period of time in media defficient in amino acid (at least, during the time course of our experiment), but grow slower. Probably this would require more time than 3 days to observe significative cell dying.<br />
<br />
These results are consistent with the chosen parameters. Moreover, the slower the death rate the bigger the area in the Parameter Space where regulation is feasable.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-10-27T03:54:25Z<p>Abush84: /* Description of strains */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|TCY 3128<br />
| style="text-align: center;" |(-H-T)<br />
|CFP <br />
|His device testing<br />
|TCY 3081<br />
| style="text-align: center;" |(-H-T)<br />
|YFP <br />
|Trp device testing<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurement of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|300px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Fluoro- igembsas2012. strains.png|650px]]<br />
|-<br />
|'''CFP Fluorescence Screening and YFP Fluorescence Screening'''<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control Mix 2: 80% CFP; 20%YFP Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP Mix 5: 40% CFP; 60%YFP Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|100px]]<br />
|[[File:Bsas2012-strains-figura2.png|100px]]<br />
|[[File:Bsas2012-strains-figura4.png|100px]]<br />
|[[File:Bsas2012-strains-figura5.png|100px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:HIS-BSAS2012.png|400px]]<br />
|}<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
|}<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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 20μg/ml, until approximately constant intensity.<br />
<br />
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 and diffusing their own Trp.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Curva.png | 250px]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
The sensitivity of this method seems to be enough to detect concentrations as low as ~0.01μg/ml, and as high as 20μg/ml, maybe more. Since our medium is 20μg/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.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC (no cells)<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
| -T<br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
| Trp/2<br />
|2.56 <br />
|2.17<br />
|-<br />
| Trp/4<br />
|3.01 <br />
|3.11<br />
|-<br />
|Trp/8<br />
|1.54 <br />
|1.55<br />
|-<br />
|Trp/16<br />
|0.393 <br />
|0.409<br />
|-<br />
|Trp/32<br />
|0.013 <br />
|0.003<br />
|-<br />
| -H<br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
| His/2 <br />
|3.68 <br />
|3.84<br />
|-<br />
| His/4<br />
|2.07 <br />
|2.00<br />
|-<br />
|His/8<br />
|1.17 <br />
|0.97<br />
|-<br />
|His/16 <br />
|0.47 <br />
|0.432<br />
|-<br />
|His/32 <br />
|0.238 <br />
|0.257<br />
|-<br />
|SC (w/cells) <br />
|4.88 <br />
|4.91<br />
|} <br />
<br />
==== Results ====<br />
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 [[Team:Buenos_Aires/Project/Model#Parameter_selection|mathematical model]], which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|250px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|250px]] <br />
|-<br />
|Images from HLU series<br />
|Images from TLU series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLU is the culture in the medium with all the required aminoacids.<br />
<br />
<br />
== Experimental determination of strains death rate==<br />
<br />
We set out to determine how long can auxotroph cells[link] survive in media that lacks both Trytophan and Histidine. These values are the '''death''' parameters for CFP and YFP strains used in our model[link]. These were taken as equal in the mathematical analysis for simplicity but now we would like to test whether this approximation is accurate.<br />
<br />
Given that our system most likely will present a lag phase until a certain amount of both AmioAcids is accumulated in the media, will the cells be viable until this occurs? This is a neccesary check of our '' system's feasability''.<br />
<br />
===== Protocol =====<br />
<br />
For this experiment we used<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-YFP.jpg|200px]]<br />
|[[File:BsAs2012-icono-CFP.jpg|200px]]<br />
|- align="center"<br />
|YFP Strain<br />
|CFP Strain<br />
|}<br />
<br />
*We set cultures of the two auxotroph strains without being transformed (YFP and CFP) in medium –HT at an initial OD of 0.01. <br />
*Each day we plated the same amount of µl of the culture and counted the number of colonies obtain in each plate. We set 3 replica of each strain.<br />
<br />
===== Result =====<br />
<br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" |Strain<br />
! scope="row" style="background: #7ac5e8" |Replica<br />
! scope="row" style="background: #7ac5e8" |Monday<br />
! scope="row" style="background: #7ac5e8" |Tuesday<br />
! scope="row" style="background: #7ac5e8" |Wednesday<br />
|-<br />
|CFP<br />
|1<br />
|260<br />
|320<br />
|285<br />
|-<br />
|CFP<br />
|2<br />
|267<br />
|314<br />
|76<br />
|-<br />
|CFP<br />
|3<br />
|413<br />
|362<br />
|278<br />
|-<br />
|YFP<br />
|1<br />
|230<br />
|316<br />
|688<br />
|-<br />
|YFP<br />
|2<br />
|291<br />
|194<br />
|524<br />
|-<br />
|YFP<br />
|3<br />
|449<br />
|344<br />
|725<br />
|}<br />
<br />
'''Table:''' Number of colonies counted per plate.<br />
<br />
We expected to see a decrease in the number of colonies because of cell death. We found that this was not the case in the experiment's time lapse. However we observed that the size of the colonies was smaller everyday as can be seen in the following pictures.<br />
<br />
[[File:Bsas2012kdeathcells.png| 500px]]<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a persistant state. In this way cells can survive for a period of time in media defficient in amino acid (at least, during the time course of our experiment), but grow slower. Probably this would require more time than 3 days to observe significative cell dying.<br />
<br />
These results are consistent with the chosen parameters. Moreover, the slower the death rate the bigger the area in the Parameter Space where regulation is feasable.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/AttributionsTeam:Buenos Aires/Attributions2012-10-27T03:47:31Z<p>Abush84: /* Attributions */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
= Attributions =<br />
<br />
* Peptides of payload devices ''PolyHb'' and ''PolyWb'' were designed by team instructors, although students got their dna sequences by retro translation, and added the parts to the registry<br />
<br />
* DNA was synthesized by IDT.<br />
<br />
* All yeast strains we worked with, and some reactants and equipment, were kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab.<br />
<br />
* '''Everything''' not mentioned explicitly before was done by student members of the team (with the advisors supervision)</div>Abush84http://2012.igem.org/File:Crossfeeding-v03-small.pngFile:Crossfeeding-v03-small.png2012-10-27T03:38:36Z<p>Abush84: </p>
<hr />
<div></div>Abush84http://2012.igem.org/Team:Buenos_AiresTeam:Buenos Aires2012-09-27T03:57:12Z<p>Abush84: </p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
= Welcome to Buenos Aires 2012 iGEM Wiki! =<br />
<br />
{|<br />
|<br />
'''What are you looking for?'''<br />
<br />
* Check the [[Team:Buenos_Aires/Safety|safety questions]] and [[ Team:Buenos_Aires/Attributions | attributions]].<br />
<br />
* Meet [[Team:Buenos_Aires/Team/Members | the team]]. You can also read a little about [[Team:Buenos_Aires/Team/BsAs | where we come from]].<br />
<br />
* Learn about [[Team:Buenos_Aires/Project | our project]] (and don't forget to check [[Team:Buenos_Aires/Project/Schemes | all the schemes]] we thought to solve the problem).<br />
<br />
* Don't miss our yeast [[Team:Buenos_Aires/Results/Strains| strains characterization]], and the [[Team:Buenos_Aires/Results/Bb1 |main biobricks and devices ]] we designed and added to the registry. You can also find information about our ''planB'' [[Team:Buenos_Aires/Results/Bb2 | backup biobrick]] ''(aka. thank-you-customs biobrick)'' .<br />
<br />
* Take a look at our [[Team:Buenos_Aires/Project/Model | mathematical model]] of a synthetic ecology. And if you dare, take a look at the [[Team:Buenos_Aires/Project/ModelAdvance | advanced model]].<br />
<br />
* What we did outside the lab ''(aka. human practices)'' to [[Team:Buenos_Aires/HP/GarageLab | teach what synBio is about]], [[Team:Buenos_Aires/HP/GarageLab | start solving local problems ]] and [[ Team:Buenos_Aires/HP/EMBO | seed SynBio in Latin America]].<br />
<br />
* Watch an [http://youtu.be/bkczB60RziU online presentation] of our team and our project.<br />
<br />
* If you understand spanish, you can also check our [http://www.youtube.com/watch?v=oEMXc6cmmgo presentation video].<br />
<br />
* Some [http://blogs.scientificamerican.com/lab-rat/2012/09/09/igem-buenos-aires-synthetic-bacterial-communities/ impact in the online media ] (Scientific American blog)<br />
<br />
|<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-The Team.png|400px]]<br />
|}<br />
<br />
|}</div>Abush84http://2012.igem.org/Team:Buenos_AiresTeam:Buenos Aires2012-09-27T03:56:25Z<p>Abush84: </p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
= Welcome to Buenos Aires 2012 iGEM Wiki! =<br />
<br />
{|<br />
|<br />
'''What are you looking for?'''<br />
<br />
* Check the [[Team:Buenos_Aires/Safety|safety questions]] and [[ Team:Buenos_Aires/Attributions | attributions]].<br />
<br />
* Meet [[Team:Buenos_Aires/Team/Members | the team]]. You can also read a little about [[Team:Buenos_Aires/Team/BsAs | where we come from]].<br />
<br />
* Learn about [[Team:Buenos_Aires/Project | our project]] (and don't forget to check [[Team:Buenos_Aires/Project/Schemes | all the schemes]] we thought to solve the problem).<br />
<br />
* Don't miss our yeast [[Team:Buenos_Aires/Results/Strains| strains characterization]], and the [[Team:Buenos_Aires/Results/Bb1 |main biobricks and devices ]] we designed and added to the registry. You can also find information about our ''planB'' [[Team:Buenos_Aires/Results/Bb2 | backup biobrick]] ''(aka. thank-you-customs biobrick)'' .<br />
<br />
* Take a look at our [[Team:Buenos_Aires/Project/Model | mathematical model]] of a synthetic ecology. And if you dare, take a look at the [[Team:Buenos_Aires/Project/ModelAdvance | advanced model]].<br />
<br />
* What we did outside the lab ''(aka. human practices)'' to [[Team:Buenos_Aires/HP/GarageLab | teach what synBio is about]], [[Team:Buenos_Aires/HP/GarageLab | start solving local problems ]] and [[ Team:Buenos_Aires/HP/EMBO | seed SynBio in Latin America]].<br />
<br />
* Watch an [http://youtu.be/bkczB60RziU online presentation] of our team and our project.<br />
<br />
* If you understand spanish, you can also check our [http://www.youtube.com/watch?v=oEMXc6cmmgo presentation video].<br />
<br />
* Some [http://blogs.scientificamerican.com/lab-rat/2012/09/09/igem-buenos-aires-synthetic-bacterial-communities/ impact in the online media ] (ScientificAmerican)<br />
<br />
|<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-The Team.png|400px]]<br />
|}<br />
<br />
|}</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Team/MembersTeam:Buenos Aires/Team/Members2012-09-27T03:53:44Z<p>Abush84: /* Advisors */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
{|<br />
| [[File:Bsas2012-banderaArgentina.png|100px|]]<br />
| align = "center" | '''We are the first Argentinian team to participate in iGEM competition, so everything is new for us! Here we are, ready to work hard on our project!'''. '''Meet us, and also check [[Team:Buenos_Aires/Team/BsAs | where we come from]] '''<br />
| [[File:Bsas2012-banderaArgentina.png|100px|]]<br />
<br />
|}<br />
<br />
== Students ==<br />
<br />
{| style="width:90%"<br />
|-<br />
| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Basas2012-Vero.jpg]]<br />
| rowspan="2" style="width: 3%; background: #009ee1;" |<br />
| style="width: 75%;" | '''Veronica Parasco - Physics Student'''<br />
|- valign="top"<br />
|I guess I like to study too much, or the stress and making summaries. I'm about to finish my Licentiate in Physics, when I started to think about what was next, I realized that several of the decision I made took me closer to other career fields. So here I am, learning biology and opening new doors.<br />
|}<br />
<br />
<br />
{| style="width:90%"<br />
|- valign="top"<br />
| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-mario.jpg|160px]]<br />
| rowspan="2" style="width: 3%; background: #009ee1;" |<br />
| style="width: 75%;" | '''Mario J. Rugiero - Chemistry and Computer Sciece Student'''<br />
|- valign="top"<br />
|Initially, the iGEM grabbed my attention through its idea of free thought and colaboration, which I consider should be general rule if we want society to progress. Even though my career choice seems remotely related to synthetic biology, curiosity moves me to learn a little bit about everything, which is never a bad idea.<br />
I'd like to see that this initiative will bring together all the synthetic biology investigation groups in Argentina because I see in this discipline an opportunity to develop and resolve many local, and very important, problems.<br />
|}<br />
<br />
<br />
{| style="width:90%"<br />
|- valign="top"<br />
| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-ale.jpg|160px]]<br />
| rowspan="2" style="width: 3%; background: #009ee1;" |<br />
| style="width: 75%;" | '''María Alejandra Parreño - Biology Student'''<br />
|- valign="top"<br />
|Within the vast field of Biology, i like ALL study subjects, but my specialty at the moment Biological Ecology of Populations (previously known as Population Genetics). Since 2009, I work in conserving the genetic variability of fruit flies which are a plague in Latin America, and I also do so with other insects with economical importance.<br />
I have another two very strong interests: on one hand, the spread of science, and on the other, the sustainable development of societies and handling of natural and biological resources. So I try to match my academic ocupations with relevant participations in congress and with activities in these two areas. I love competition, innovation, and challenges, which is what attracted me to participate in iGEM and test my abilities. I see an unexplored synthetic biology field in Argentina, with a great potential to solve scientific and social problems, using wits and creativity as the main tools. As a first iGEM group, we want to impulse these ideas into reality and start off with the right foot!<br />
|}<br />
<br />
<br />
{| style="width:90%"<br />
|- valign="top"<br />
| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-lucho.jpg|160px]]<br />
| rowspan="2" style="width: 3%; background: #009ee1;" |<br />
| style="width: 75%;" | '''Luciano G. Morosi- Biology Student'''<br />
|- valign="top"<br />
| I am currently going through my last year, and i've been an intern in an investigation laboratory. From a very young age, I was passionate and interested in natural sciences, and I decided to study biology because you must have knowledge in all sciences to be able to understand it. In this way, my interest in synthetic biology comes from the fact that it is multidisciplinary, and I'm immensely attracted to the idea of being able to create biological systems – or based in biological parts- that are innovative, which carry out specifically designed actions, using and creating standarized and combined parts. I'm not only attracted to science, but also literature, theater, music and sports: for a very long time I participated in gymnastics and swimming, and I still do the latter. I love to write, I feel comfortable writing social, political and cultural papers, and also stories.<br />
|}<br />
<br />
<br />
{| style="width:90%"<br />
|- valign="top"<br />
| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-manugimenez.jpg|160px]]<br />
| rowspan="2" style="width: 3%; background: #009ee1;" |<br />
| style="width: 75%;" | '''Manuel Giménez - Computer Science Student'''<br />
|- valign="top"<br />
| I'm writing my licentiate thesis on building a tool to automatically reason about regulations. Yes yes, nothing relates to biology. Weird, right? Nevertheless, I've always had an interest for this discipline, mainly molecular biology. When I found out about the existance of iGEM – a couple of months ago- I said ''we have to assemble a team in Argentina''. Coincidence or not, a week later I got an email inviting me to be a part of the first Argentinian iGEM team, and now I find myself taking my first steps in synthetic biology as a member of iGEM BsAs. I'm mainly interested in the engineering vision that synthetic biology has, and I believe from the computer science standpoint, I have several ideas I can bring to this new subject. I love scientific dissemination and teaching; I consider myself a straightforward communicator, and my natural way of working is in groups. I'm part of a political movements in my university, and I try to make my passing through this world the most transforming and engaging possible.<br />
|}<br />
<br />
== Advisors ==<br />
<br />
{| style="width:90%"<br />
|- valign="top"<br />
| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-alan.jpg|160px]]<br />
| rowspan="2" style="width: 3%; background: #004a99;" |<br />
| style="width: 75%;" | '''Alan Bush - M.Sc. in Biology'''<br />
|- valign="top"<br />
|I'm M. Sc. in Biology and I'm currently doing my Ph. D. in Biology. My studies are in the field of "systems biology", an area which attempts to give a more quantitative and integrative approach to molecular biology, through the use of mathematic modeling tools. It is related to synthetic biology since it uses the same kind of tools and model organisms. However, the focus is radically different; while systems biology aims to understand how the cells work, the main objective of synthetic biology is to design and produce "biological devices" with a given behavior. My main motivation for participating as advisor for UBA's iGEM team is precisely this approach switch. I'm fascinated with the idea of using our knowledge to develop useful devices which can help to solve concrete problems.<br />
|}<br />
<br />
<br />
{| style="width:90%"<br />
|- valign="top"<br />
| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-german.jpg|160px]]<br />
| rowspan="2" style="width: 3%; background: #004a99;" |<br />
| style="width: 75%;" | '''German Sabio - M.Sc. in Biology'''<br />
|- valign="top"<br />
|I'm an extremely curious person, and I've always had a passion for all kind of "bugs" (a highly academic and very complex concept, which includes everything from cell and virus to mammal and aliens) and how "life" works. A M. Sc. in Biology didn't gave me the answer just yet, but kept my curiosity appeased for some years and gave me a big ammount of tools to keep asking new questions.<br />
Currently, I work on a branch of biology dedicated to living being's development: how, from a single cell, or from a group of similar cells, a differentiated organism gets developed. My Ph. D. discipline is systems biology, which basically studies several biology areas looking for math patterns and predicting (or, actually, modelling) different living systems. Synthetic biology would be the other face of the same coin: while systems biology tries to find and define mechanisms in nature to understand how they work, synthetic biology tries to reproduce or generate new systems with a predetermined function.<br />
I'm very interested in this chance to take part on an iGEM team, not just for the ammount of tools it represents, but because it's a good and interesting experience to start a group and discuss and work together.<br />
|}<br />
<br />
== Instructors==<br />
<br />
{| style="width:90%"<br />
|- valign="top"<br />
| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-nacho.jpg|160px]]<br />
| rowspan="2" style="width: 3%; background: #0B2161;" |<br />
| style="width: 75%;" | '''Ignacio E. Sánchez - M.Sc. in Chemistry and Ph.D. in Biophysics'''<br />
|- valign="top"<br />
|I'm a spanish bioinformatics specialist, currently living in Argentina. Before, I was an in training biophysicist on northern Europe, and before, a chemist, fascinated by biological molecules. Day by day, I study oncogenic virus in the Department of Biological Chemistry's Protein's Physiology Laboratory.<br />
I think synthetic biology is an excellent chance for developing countries to acquire new capabilities in the subject of biotechnology. Because of that reason, I joined Dr. Nadra in 2011 to promote the formation of synthetic biologists in Argentina. By now, the experience is being highly fun and rewarding.<br />
|}<br />
<br />
<br />
{| style="width:90%"<br />
|- valign="top"<br />
| rowspan="2" style="width: 20%; text-align: center;" | [[Image:Bsas2012-alenadra.jpg|160px]]<br />
| rowspan="2" style="width: 3%; background: #0B2161;" |<br />
| style="width: 75%;" | '''Alejandro D. Nadra - M.Sc. in Biology and Ph.D. in Chemistry'''<br />
|- valign="top"<br />
|I'm M. Sc. in Biology (2001) and Ph. D. in Chemistry (2005). My subjects of interest are protein's structure/function/folding and their interaction with nucleic acids. Also hemoproteins, evolution and synthetic biology, and others. I did a post-doc with the molecular modelling group in the FCEyN and another one in the systems biology program of the Genomic Regulation Center from Barcelona.<br />
I started to work as teacher in the FCEyN in 2000 and I'm currently Assistant Professor in the Department of Biological Chemistry and researcher at CONICET. I'm also a member of the Structural Biochemistry Laboratory, Department of Biological Chemistry.<br />
Given the lack of Synthetic Biology in Argentina, we are promoting the area with Ignacio Sánchez since 2011. For this effort, we organized the first post-graduate course in the subject in 2011, and conducted a course with featured international referents in April 2012. I'm convinced that from my place and given my formation, I can boost Synthetic Biology and begin formation of pure strain Synthetic Biologists. I believe the iGEM competitions are an excellente tool for the education and motivation of students. I'm convinced that from Argentina we can contribute to the subject and be on pair with teams from everywhere in the world. Even though we count on less resources and funds, this lack is compensated with a huge motivation and creativity.<br />
|}</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-09-27T03:45:31Z<p>Abush84: /* Results */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurment of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|300px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Yfp.png|450px]]<br />
| align="center" | [[File:Bsas2012-strains-grafico2.png|500px]]<br />
|-<br />
|YFP Fluorescence Screening<br />
|CFP Fluorescence Screening<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control<br />
<br />
Mix 2: 80% CFP; 20%YFP<br />
<br />
Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP<br />
<br />
Mix 5: 40% CFP; 60%YFP<br />
<br />
Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|300px]]<br />
|[[File:Bsas2012-strains-figura2.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
|}<br />
<br />
{| class="wikitable"<br />
|-<br />
|[[File:Bsas2012-strains-figura4.png|300px]]<br />
|[[File:Bsas2012-strains-figura5.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strain-Grafale2.png|800px]]<br />
|}<br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Grafale1.png|400px]]<br />
|<!--column1-->[[File:Bsas2012-strains-grafico5.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
|}<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium and Basal Production ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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.<br />
<br />
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.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Trpmario.png]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
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.<br />
<br />
Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC (no cells)<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
| -T<br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
| Trp/2<br />
|2.56 <br />
|2.17<br />
|-<br />
| Trp/4<br />
|3.01 <br />
|3.11<br />
|-<br />
|Trp/8<br />
|1.54 <br />
|1.55<br />
|-<br />
|Trp/16<br />
|0.393 <br />
|0.409<br />
|-<br />
|Trp/32<br />
|0.013 <br />
|0.003<br />
|-<br />
| -H<br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
| His/2 <br />
|3.68 <br />
|3.84<br />
|-<br />
| His/4<br />
|2.07 <br />
|2.00<br />
|-<br />
|His/8<br />
|1.17 <br />
|0.97<br />
|-<br />
|His/16 <br />
|0.47 <br />
|0.432<br />
|-<br />
|His/32 <br />
|0.238 <br />
|0.257<br />
|-<br />
|SC (w/cells) <br />
|4.88 <br />
|4.91<br />
|} <br />
<br />
==== Results ====<br />
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 [[Team:Buenos_Aires/Project/Model#Parameter_selection|mathematical model]], which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|500px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|500px]] <br />
|-<br />
|Images from HLU series<br />
|Images from TLU series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLU is the culture in the medium with all the required aminoacids.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-09-27T03:44:13Z<p>Abush84: /* Results */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurment of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|450px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Yfp.png|450px]]<br />
| align="center" | [[File:Bsas2012-strains-grafico2.png|500px]]<br />
|-<br />
|YFP Fluorescence Screening<br />
|CFP Fluorescence Screening<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control<br />
<br />
Mix 2: 80% CFP; 20%YFP<br />
<br />
Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP<br />
<br />
Mix 5: 40% CFP; 60%YFP<br />
<br />
Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|300px]]<br />
|[[File:Bsas2012-strains-figura2.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
|}<br />
<br />
{| class="wikitable"<br />
|-<br />
|[[File:Bsas2012-strains-figura4.png|300px]]<br />
|[[File:Bsas2012-strains-figura5.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strain-Grafale2.png|800px]]<br />
|}<br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Grafale1.png|400px]]<br />
|<!--column1-->[[File:Bsas2012-strains-grafico5.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium and Basal Production ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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.<br />
<br />
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.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Trpmario.png]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
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.<br />
<br />
Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC (no cells)<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
| -T<br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
| Trp/2<br />
|2.56 <br />
|2.17<br />
|-<br />
| Trp/4<br />
|3.01 <br />
|3.11<br />
|-<br />
|Trp/8<br />
|1.54 <br />
|1.55<br />
|-<br />
|Trp/16<br />
|0.393 <br />
|0.409<br />
|-<br />
|Trp/32<br />
|0.013 <br />
|0.003<br />
|-<br />
| -H<br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
| His/2 <br />
|3.68 <br />
|3.84<br />
|-<br />
| His/4<br />
|2.07 <br />
|2.00<br />
|-<br />
|His/8<br />
|1.17 <br />
|0.97<br />
|-<br />
|His/16 <br />
|0.47 <br />
|0.432<br />
|-<br />
|His/32 <br />
|0.238 <br />
|0.257<br />
|-<br />
|SC (w/cells) <br />
|4.88 <br />
|4.91<br />
|} <br />
<br />
==== Results ====<br />
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 [[Team:Buenos_Aires/Project/Model#Paramter_selection|mathematical model]], which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|500px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|500px]] <br />
|-<br />
|Images from HLU series<br />
|Images from TLU series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLU is the culture in the medium with all the required aminoacids.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-09-27T03:41:58Z<p>Abush84: /* Growth dependence on the Trp and His concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurment of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|450px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Yfp.png|450px]]<br />
| align="center" | [[File:Bsas2012-strains-grafico2.png|500px]]<br />
|-<br />
|YFP Fluorescence Screening<br />
|CFP Fluorescence Screening<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control<br />
<br />
Mix 2: 80% CFP; 20%YFP<br />
<br />
Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP<br />
<br />
Mix 5: 40% CFP; 60%YFP<br />
<br />
Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|300px]]<br />
|[[File:Bsas2012-strains-figura2.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
|}<br />
<br />
{| class="wikitable"<br />
|-<br />
|[[File:Bsas2012-strains-figura4.png|300px]]<br />
|[[File:Bsas2012-strains-figura5.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strain-Grafale2.png|800px]]<br />
|}<br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Grafale1.png|400px]]<br />
|<!--column1-->[[File:Bsas2012-strains-grafico5.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium and Basal Production ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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.<br />
<br />
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.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Trpmario.png]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
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.<br />
<br />
Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC (no cells)<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
| -T<br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
| Trp/2<br />
|2.56 <br />
|2.17<br />
|-<br />
| Trp/4<br />
|3.01 <br />
|3.11<br />
|-<br />
|Trp/8<br />
|1.54 <br />
|1.55<br />
|-<br />
|Trp/16<br />
|0.393 <br />
|0.409<br />
|-<br />
|Trp/32<br />
|0.013 <br />
|0.003<br />
|-<br />
| -H<br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
| His/2 <br />
|3.68 <br />
|3.84<br />
|-<br />
| His/4<br />
|2.07 <br />
|2.00<br />
|-<br />
|His/8<br />
|1.17 <br />
|0.97<br />
|-<br />
|His/16 <br />
|0.47 <br />
|0.432<br />
|-<br />
|His/32 <br />
|0.238 <br />
|0.257<br />
|-<br />
|SC (w/cells) <br />
|4.88 <br />
|4.91<br />
|} <br />
<br />
==== Results ====<br />
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 [[Team:Buenos_Aires/Project/Model#Paramter_selection|mathematical model]], which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|500px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|500px]] <br />
|-<br />
|Images from HLU series<br />
|Images from TLU series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLU is the culture in the medium with all the required aminoacids.<br />
<br />
S(Number) are the serial dilutions of HTLC with medium that lacks Histidine (HLC) and Tryptophane (TLC).<br />
<br />
HLU and TLU are mediums without cells.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-09-27T03:40:08Z<p>Abush84: /* Growth dependence on the Trp and His concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurment of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|450px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Yfp.png|450px]]<br />
| align="center" | [[File:Bsas2012-strains-grafico2.png|500px]]<br />
|-<br />
|YFP Fluorescence Screening<br />
|CFP Fluorescence Screening<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control<br />
<br />
Mix 2: 80% CFP; 20%YFP<br />
<br />
Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP<br />
<br />
Mix 5: 40% CFP; 60%YFP<br />
<br />
Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|300px]]<br />
|[[File:Bsas2012-strains-figura2.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
|}<br />
<br />
{| class="wikitable"<br />
|-<br />
|[[File:Bsas2012-strains-figura4.png|300px]]<br />
|[[File:Bsas2012-strains-figura5.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strain-Grafale2.png|800px]]<br />
|}<br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Grafale1.png|400px]]<br />
|<!--column1-->[[File:Bsas2012-strains-grafico5.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium and Basal Production ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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.<br />
<br />
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.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Trpmario.png]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
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.<br />
<br />
Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC (no cells)<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
| -T<br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
| Trp/2<br />
|2.56 <br />
|2.17<br />
|-<br />
| Trp/4<br />
|3.01 <br />
|3.11<br />
|-<br />
|Trp/8<br />
|1.54 <br />
|1.55<br />
|-<br />
|Trp/16<br />
|0.393 <br />
|0.409<br />
|-<br />
|Trp/32<br />
|0.013 <br />
|0.003<br />
|-<br />
| -H<br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
| His/2 <br />
|3.68 <br />
|3.84<br />
|-<br />
| His/4<br />
|2.07 <br />
|2.00<br />
|-<br />
|His/8<br />
|1.17 <br />
|0.97<br />
|-<br />
|His/16 <br />
|0.47 <br />
|0.432<br />
|-<br />
|His/32 <br />
|0.238 <br />
|0.257<br />
|-<br />
|SC (w/cells) <br />
|4.88 <br />
|4.91<br />
|} <br />
<br />
==== Results ====<br />
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 [[Team:Buenos_Aires/Results/Model|mathematical model]], which can be used to estimate the secretion rate of each aminoacid needed to get effective crossfeeding. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|500px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|500px]] <br />
|-<br />
|Images from HLU series<br />
|Images from TLU series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLU is the culture in the medium with all the required aminoacids.<br />
<br />
S(Number) are the serial dilutions of HTLC with medium that lacks Histidine (HLC) and Tryptophane (TLC).<br />
<br />
HLU and TLU are mediums without cells.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-09-27T03:30:51Z<p>Abush84: /* Measurement of Trp in medium and Basal Production */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurment of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|450px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Yfp.png|450px]]<br />
| align="center" | [[File:Bsas2012-strains-grafico2.png|500px]]<br />
|-<br />
|YFP Fluorescence Screening<br />
|CFP Fluorescence Screening<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control<br />
<br />
Mix 2: 80% CFP; 20%YFP<br />
<br />
Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP<br />
<br />
Mix 5: 40% CFP; 60%YFP<br />
<br />
Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|300px]]<br />
|[[File:Bsas2012-strains-figura2.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
|}<br />
<br />
{| class="wikitable"<br />
|-<br />
|[[File:Bsas2012-strains-figura4.png|300px]]<br />
|[[File:Bsas2012-strains-figura5.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strain-Grafale2.png|800px]]<br />
|}<br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Grafale1.png|400px]]<br />
|<!--column1-->[[File:Bsas2012-strains-grafico5.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium and Basal Production ==<br />
<br />
To check the efectiveness of our biobricks, we must first determine the ammount of tryptophan secreted 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.<br />
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.<br />
<br />
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.<br />
<br />
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.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Trpmario.png]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
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.<br />
<br />
Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
|HLC <br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
|S1 HLC (1:10) <br />
|0,256 <br />
|0,217<br />
|-<br />
|S2 HLC (1:10) <br />
|0,301 <br />
|0,311<br />
|-<br />
|S3 HLC (1:10) <br />
|0,154 <br />
|0,155<br />
|-<br />
|S4 HLC <br />
|0,393 <br />
|0,409<br />
|-<br />
|S5 HLC <br />
|0,013 <br />
|0,003<br />
|-<br />
|TLC <br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
|S1 TLC (1:10) <br />
|0,368 <br />
|0,384<br />
|-<br />
|S2 TLC (1:10) <br />
|0,207 <br />
|0,2<br />
|-<br />
|S3 TLC (1:10) <br />
|0,117 <br />
|0,097<br />
|-<br />
|S4 TLC <br />
|0,47 <br />
|0,432<br />
|-<br />
|S5 TLC <br />
|0,238 <br />
|0,257<br />
|-<br />
|HTLC (1:10) <br />
|0,488 <br />
|0,491<br />
|} <br />
<br />
==== Results ====<br />
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. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|500px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|500px]] <br />
|-<br />
|Images from HLC series<br />
|Images from TLC series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLC is the culture in the medium with all the required aminoacids.<br />
<br />
S(Number) are the serial dilutions of HTLC with medium that lacks Histidine (HLC) and Tryptophane (TLC).<br />
<br />
HLC and TLC are mediums without cells.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-09-27T03:26:00Z<p>Abush84: /* Coculture in Agar and Revertant mutation control */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurment of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|450px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Yfp.png|450px]]<br />
| align="center" | [[File:Bsas2012-strains-grafico2.png|500px]]<br />
|-<br />
|YFP Fluorescence Screening<br />
|CFP Fluorescence Screening<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control<br />
<br />
Mix 2: 80% CFP; 20%YFP<br />
<br />
Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP<br />
<br />
Mix 5: 40% CFP; 60%YFP<br />
<br />
Mix 6: 20% CFP; 80%YFP<br />
<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|900px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|300px]]<br />
|[[File:Bsas2012-strains-figura2.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
|}<br />
<br />
{| class="wikitable"<br />
|-<br />
|[[File:Bsas2012-strains-figura4.png|300px]]<br />
|[[File:Bsas2012-strains-figura5.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strain-Grafale2.png|800px]]<br />
|}<br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Grafale1.png|400px]]<br />
|<!--column1-->[[File:Bsas2012-strains-grafico5.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
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. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
<br />
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.<br />
<br />
== Measurement of Trp in medium and Basal Production ==<br />
<br />
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.<br />
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.<br />
<br />
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.<br />
<br />
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.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Trpmario.png]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
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.<br />
<br />
Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
|HLC <br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
|S1 HLC (1:10) <br />
|0,256 <br />
|0,217<br />
|-<br />
|S2 HLC (1:10) <br />
|0,301 <br />
|0,311<br />
|-<br />
|S3 HLC (1:10) <br />
|0,154 <br />
|0,155<br />
|-<br />
|S4 HLC <br />
|0,393 <br />
|0,409<br />
|-<br />
|S5 HLC <br />
|0,013 <br />
|0,003<br />
|-<br />
|TLC <br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
|S1 TLC (1:10) <br />
|0,368 <br />
|0,384<br />
|-<br />
|S2 TLC (1:10) <br />
|0,207 <br />
|0,2<br />
|-<br />
|S3 TLC (1:10) <br />
|0,117 <br />
|0,097<br />
|-<br />
|S4 TLC <br />
|0,47 <br />
|0,432<br />
|-<br />
|S5 TLC <br />
|0,238 <br />
|0,257<br />
|-<br />
|HTLC (1:10) <br />
|0,488 <br />
|0,491<br />
|} <br />
<br />
==== Results ====<br />
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. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|500px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|500px]] <br />
|-<br />
|Images from HLC series<br />
|Images from TLC series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLC is the culture in the medium with all the required aminoacids.<br />
<br />
S(Number) are the serial dilutions of HTLC with medium that lacks Histidine (HLC) and Tryptophane (TLC).<br />
<br />
HLC and TLC are mediums without cells.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-09-27T03:17:54Z<p>Abush84: /* Coculture in Agar and Revertant mutation control */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurment of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|450px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Yfp.png|450px]]<br />
| align="center" | [[File:Bsas2012-strains-grafico2.png|500px]]<br />
|-<br />
|YFP Fluorescence Screening<br />
|CFP Fluorescence Screening<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control<br />
<br />
Mix 2: 80% CFP; 20%YFP<br />
<br />
Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP<br />
<br />
Mix 5: 40% CFP; 60%YFP<br />
<br />
Mix 6: 20% CFP; 80%YFP<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|800px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|300px]]<br />
|[[File:Bsas2012-strains-figura2.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
|}<br />
<br />
{| class="wikitable"<br />
|-<br />
|[[File:Bsas2012-strains-figura4.png|300px]]<br />
|[[File:Bsas2012-strains-figura5.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strain-Grafale2.png|800px]]<br />
|}<br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Grafale1.png|400px]]<br />
|<!--column1-->[[File:Bsas2012-strains-grafico5.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
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. <br />
<br />
We used petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
At the same time, 3 controls (one for each strain) were carried in YPD complete medium to check the viability of each strain separately. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Lawn (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected, as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
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 amino acids.<br />
<br />
== Measurement of Trp in medium and Basal Production ==<br />
<br />
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.<br />
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.<br />
<br />
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.<br />
<br />
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.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Trpmario.png]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
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.<br />
<br />
Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
|HLC <br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
|S1 HLC (1:10) <br />
|0,256 <br />
|0,217<br />
|-<br />
|S2 HLC (1:10) <br />
|0,301 <br />
|0,311<br />
|-<br />
|S3 HLC (1:10) <br />
|0,154 <br />
|0,155<br />
|-<br />
|S4 HLC <br />
|0,393 <br />
|0,409<br />
|-<br />
|S5 HLC <br />
|0,013 <br />
|0,003<br />
|-<br />
|TLC <br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
|S1 TLC (1:10) <br />
|0,368 <br />
|0,384<br />
|-<br />
|S2 TLC (1:10) <br />
|0,207 <br />
|0,2<br />
|-<br />
|S3 TLC (1:10) <br />
|0,117 <br />
|0,097<br />
|-<br />
|S4 TLC <br />
|0,47 <br />
|0,432<br />
|-<br />
|S5 TLC <br />
|0,238 <br />
|0,257<br />
|-<br />
|HTLC (1:10) <br />
|0,488 <br />
|0,491<br />
|} <br />
<br />
==== Results ====<br />
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. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|500px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|500px]] <br />
|-<br />
|Images from HLC series<br />
|Images from TLC series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLC is the culture in the medium with all the required aminoacids.<br />
<br />
S(Number) are the serial dilutions of HTLC with medium that lacks Histidine (HLC) and Tryptophane (TLC.<br />
<br />
HLC and TLC are mediums without cells.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-09-27T03:13:25Z<p>Abush84: /* Results */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurment of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|450px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Yfp.png|450px]]<br />
| align="center" | [[File:Bsas2012-strains-grafico2.png|500px]]<br />
|-<br />
|YFP Fluorescence Screening<br />
|CFP Fluorescence Screening<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control<br />
<br />
Mix 2: 80% CFP; 20%YFP<br />
<br />
Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP<br />
<br />
Mix 5: 40% CFP; 60%YFP<br />
<br />
Mix 6: 20% CFP; 80%YFP<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|800px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|300px]]<br />
|[[File:Bsas2012-strains-figura2.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
|}<br />
<br />
{| class="wikitable"<br />
|-<br />
|[[File:Bsas2012-strains-figura4.png|300px]]<br />
|[[File:Bsas2012-strains-figura5.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strain-Grafale2.png|800px]]<br />
|}<br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Grafale1.png|400px]]<br />
|<!--column1-->[[File:Bsas2012-strains-grafico5.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
Through this experiment we aim to quantify the rate of revertants of each strain. <br />
<br />
We used Petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
At the same time, 3 controls (one for each strain) were carried in YPD complete medium to check the viability of each strain separately. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Grass (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected as well as the viability of a control positive strain in the –H-T medium. <br />
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 may as well be of the same. <br />
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 now 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.<br />
<br />
== Measurement of Trp in medium and Basal Production ==<br />
<br />
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.<br />
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.<br />
<br />
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.<br />
<br />
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.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Trpmario.png]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
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.<br />
<br />
Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
|HLC <br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
|S1 HLC (1:10) <br />
|0,256 <br />
|0,217<br />
|-<br />
|S2 HLC (1:10) <br />
|0,301 <br />
|0,311<br />
|-<br />
|S3 HLC (1:10) <br />
|0,154 <br />
|0,155<br />
|-<br />
|S4 HLC <br />
|0,393 <br />
|0,409<br />
|-<br />
|S5 HLC <br />
|0,013 <br />
|0,003<br />
|-<br />
|TLC <br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
|S1 TLC (1:10) <br />
|0,368 <br />
|0,384<br />
|-<br />
|S2 TLC (1:10) <br />
|0,207 <br />
|0,2<br />
|-<br />
|S3 TLC (1:10) <br />
|0,117 <br />
|0,097<br />
|-<br />
|S4 TLC <br />
|0,47 <br />
|0,432<br />
|-<br />
|S5 TLC <br />
|0,238 <br />
|0,257<br />
|-<br />
|HTLC (1:10) <br />
|0,488 <br />
|0,491<br />
|} <br />
<br />
==== Results ====<br />
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. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|500px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|500px]] <br />
|-<br />
|Images from HLC series<br />
|Images from TLC series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLC is the culture in the medium with all the required aminoacids.<br />
<br />
S(Number) are the serial dilutions of HTLC with medium that lacks Histidine (HLC) and Tryptophane (TLC.<br />
<br />
HLC and TLC are mediums without cells.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-09-27T03:11:16Z<p>Abush84: /* Coculture in Agar and Revertant mutation control */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurment of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|450px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Yfp.png|450px]]<br />
| align="center" | [[File:Bsas2012-strains-grafico2.png|500px]]<br />
|-<br />
|YFP Fluorescence Screening<br />
|CFP Fluorescence Screening<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control<br />
<br />
Mix 2: 80% CFP; 20%YFP<br />
<br />
Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP<br />
<br />
Mix 5: 40% CFP; 60%YFP<br />
<br />
Mix 6: 20% CFP; 80%YFP<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|800px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|300px]]<br />
|[[File:Bsas2012-strains-figura2.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
|}<br />
<br />
{| class="wikitable"<br />
|-<br />
|[[File:Bsas2012-strains-figura4.png|300px]]<br />
|[[File:Bsas2012-strains-figura5.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strain-Grafale2.png|800px]]<br />
|}<br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Grafale1.png|400px]]<br />
|<!--column1-->[[File:Bsas2012-strains-grafico5.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
Through this experiment we aim to quantify the rate of revertants of each strain. <br />
<br />
We used Petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
At the same time, 3 controls (one for each strain) were carried in YPD complete medium to check the viability of each strain separately. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Grass (10^6 cells) <br />
! scope="row" style="background: #7ac5e8"|Seed (10^2 cells) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
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.<br />
<br />
== Measurement of Trp in medium and Basal Production ==<br />
<br />
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.<br />
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.<br />
<br />
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.<br />
<br />
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.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Trpmario.png]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
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.<br />
<br />
Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
|HLC <br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
|S1 HLC (1:10) <br />
|0,256 <br />
|0,217<br />
|-<br />
|S2 HLC (1:10) <br />
|0,301 <br />
|0,311<br />
|-<br />
|S3 HLC (1:10) <br />
|0,154 <br />
|0,155<br />
|-<br />
|S4 HLC <br />
|0,393 <br />
|0,409<br />
|-<br />
|S5 HLC <br />
|0,013 <br />
|0,003<br />
|-<br />
|TLC <br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
|S1 TLC (1:10) <br />
|0,368 <br />
|0,384<br />
|-<br />
|S2 TLC (1:10) <br />
|0,207 <br />
|0,2<br />
|-<br />
|S3 TLC (1:10) <br />
|0,117 <br />
|0,097<br />
|-<br />
|S4 TLC <br />
|0,47 <br />
|0,432<br />
|-<br />
|S5 TLC <br />
|0,238 <br />
|0,257<br />
|-<br />
|HTLC (1:10) <br />
|0,488 <br />
|0,491<br />
|} <br />
<br />
==== Results ====<br />
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. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|500px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|500px]] <br />
|-<br />
|Images from HLC series<br />
|Images from TLC series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLC is the culture in the medium with all the required aminoacids.<br />
<br />
S(Number) are the serial dilutions of HTLC with medium that lacks Histidine (HLC) and Tryptophane (TLC.<br />
<br />
HLC and TLC are mediums without cells.</div>Abush84http://2012.igem.org/Team:Buenos_Aires/Results/StrainsTeam:Buenos Aires/Results/Strains2012-09-27T03:08:19Z<p>Abush84: /* Auxotrophy confirmation */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
<br />
== Description of strains ==<br />
<br />
Through our experiments we worked with the following strains kindly provided by [http://www.ifibyne.fcen.uba.ar/new/temas-de-investigacion/laboratorio-de-fisiologia-y-biologia-molecular-lfbm/biologia-de-sistemas/dr-alejandro-colman-lerner/ Alejandro Colman-Lerner's] Lab: <br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8" | Strain ID<br />
! scope="row" style="background: #7ac5e8" |Relevant Auxotrophies<br />
! scope="row" style="background: #7ac5e8" |Fluorescence<br />
! scope="row" style="background: #7ac5e8" |Used as<br />
|-<br />
|TCY 3043<br />
| style="text-align: center;" |(-H-T)<br />
|No fluorescence<br />
|Negative control<br />
|-<br />
|TCY 3190<br />
| style="text-align: center;" |(+H-T)<br />
|YFP + (Induced CFP)<br />
|For coculture<br />
|-<br />
|TCY 3265<br />
| style="text-align: center;" |(-H+T)<br />
|CFP<br />
|For coculture<br />
|-<br />
|TCY 3154<br />
| style="text-align: center;" |(+H+T)<br />
|CFP +(induced YFP)<br />
|Positive Control<br />
|}<br />
| rowspan="2" style="text-align: center;" |<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-figura1.jpg|300px]]<br />
|}<br />
|- valign="top"<br />
|<br />
<br />
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.<br />
<br />
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)<br />
|}<br />
<br />
== Measurment of strains fluorescence ==<br />
<br />
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. <br />
<br />
'''Reference graph'''<br />
Image: YFP and CFP Emission and Absorption Spectra. Obtained from http://flowcyt.salk.edu/fluo.html<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Refefluro.png|450px]]<br />
|}<br />
<br />
<br />
'''Results'''<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-strains-Yfp.png|450px]]<br />
| align="center" | [[File:Bsas2012-strains-grafico2.png|500px]]<br />
|-<br />
|YFP Fluorescence Screening<br />
|CFP Fluorescence Screening<br />
|}<br />
<br />
When measuring YFP Strain 3281, we can see a clear peak around 530 while when measuring CFP Strain 3265, we can see a clear peak around 500, as expected.<br />
<br />
<br />
<br />
'''Discussion'''<br />
<br />
We were able to measure fluorescence in strains 3281 and 3265 using the spectrofluorometer. However, we considered it would not be precise enough for the purposes of measuring cocultures at different proportions. We also noticed a high background noise produced by dead yeast cells at high concentrations, which would make it possible to measure in this way only at a short range of OD while the culture is at exponential phase.<br />
<br />
== Screening of strain proportion ==<br />
<br />
A more precise way of measuring the proportion of the strains, is with a epifluorescence microscope.<br />
<br />
We mixed strains 3281 (expresses YFP) and 3265 (expresses CFP) in different proportions and analized the images obtained in the microscope, where we counted cells with different fluorescences. We also did a negative control with a non fluorescent strain (TCY 379). <br />
<br />
'''Description of Mixtures'''<br />
<br />
Mix 1: Negative Control<br />
<br />
Mix 2: 80% CFP; 20%YFP<br />
<br />
Mix 3: 60% CFP; 40%YFP<br />
<br />
Mix 4: 50% CFP; 50%YFP<br />
<br />
Mix 5: 40% CFP; 60%YFP<br />
<br />
Mix 6: 20% CFP; 80%YFP<br />
<br />
'''Results'''<br />
<br />
<br />
<br />
{| style="width: 100%"<br />
| align="center" | [[File:Montage-annotated.jpg|800px]]<br />
|-<br />
| style="text-align: center;" | Mixtures showing YFP and CFP fluorescence. <br />
|}<br />
<br />
<br />
<br />
As shown by images 1-6, cells showing different fluorescences can be count and distinguished from each other in a mixture of strains, and this could be used to measure strains proportion in a coculture. <br />
<br />
<br />
'''Counting of cells'''<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Fluorescence<br />
|Mix 1<br />
|Mix 2<br />
|Mix 3<br />
|Mix 4<br />
|Mix 5<br />
|Mix 6<br />
|-<br />
|YFP<br />
|0 <br />
|23 <br />
|67 <br />
|115 <br />
|135 <br />
|110<br />
|-<br />
|CFP<br />
|0* <br />
|235 <br />
|82 <br />
|107 <br />
|99 <br />
|78<br />
|}<br />
<br />
The table shows the number of cells counted by expression of fluorescence YFP and CFP in the different mixtures 1-6. I can be observed that the amount of cells is near the proportion stablished by OD measures when preparing the mixtures. This results confirms that epifluorescence measures are reliable and suitable for our research.<br />
<br />
== Auxotrophy confirmation ==<br />
<br />
<br />
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). <br />
We observed differential growth according to expected due to the description of each strain in point a)<br />
<br />
{| class="wikitable" <br />
|+ Auxotrophy check<br />
|-<br />
|[[File:Bsas2012-strains-figura3.jpg|300px]]<br />
|[[File:Bsas2012-strains-figura2.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium complete<br />
| style="text-align: center;" | Medium without H<br />
|}<br />
<br />
{| class="wikitable"<br />
|-<br />
|[[File:Bsas2012-strains-figura4.png|300px]]<br />
|[[File:Bsas2012-strains-figura5.png|300px]]<br />
|-<br />
| style="text-align: center;" | Medium without T<br />
| style="text-align: center;" | Medium without H and T<br />
|}<br />
<br />
We observed all the strains grew in the SC plate (top left) and only 3154 (+H+T) grew in the -H-T plate (bottom right). In the -T plate (bottom left), only those strains able to synthesize T grew (3265 and 3154) and in the -H plate (top right) 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.<br />
<br />
== Coculture in liquid medium ==<br />
<br />
We used for these experiment TCY3190(H+T-) and TCY3265(H-T+)<br />
Positive control: TCY3154 (H+T+) and negative control TCY3043(H-T-)<br />
<br />
==== At different initial OD and proportions ====<br />
<br />
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). <br />
<br />
<br />
{| class="wikitable"<br />
|+ Coculture at different initial OD and proportions (Days 0 and 1)<br />
! scope="row" style="background: #7ac5e8" | Coculture Proportion (H+T-):(H-T+) <br />
! scope="row" style="background: #7ac5e8" |Initial OD(t=0) <br />
! scope="row" style="background: #7ac5e8"|OD1 (t=1) <br />
! scope="row" style="background: #7ac5e8"|OD2 (t=1) <br />
! scope="row" style="background: #7ac5e8"|dilution used for measure t=1 <br />
! scope="row" style="background: #7ac5e8"|Mean OD <br />
! scope="row" style="background: #7ac5e8"|Growth Factor<br />
|-<br />
|01:01 <br />
|0,25 <br />
|0,32 <br />
|0,314 <br />
|10 <br />
|3,17 <br />
|12,68<br />
|-<br />
|09:01 <br />
|0,25 <br />
|0,148 <br />
|0,144 <br />
|10 <br />
|1,46 <br />
|5,84<br />
|-<br />
|01:09 <br />
|0,25 <br />
|0,138 <br />
|0,189 <br />
|10 <br />
|1,635 <br />
|6,54<br />
|-<br />
|01:01 <br />
|0,1 <br />
|0,109 <br />
|0,169 <br />
|10 <br />
|1,39 <br />
|13,9<br />
|-<br />
|09:01 <br />
|0,1 <br />
|0,04 <br />
|0,045 <br />
|10 <br />
|0,425 <br />
|4,25<br />
|-<br />
|01:09 <br />
|0,1 <br />
|0,067 <br />
|0,053 <br />
|10 <br />
|0,6 <br />
|6<br />
|-<br />
|01:01 <br />
|0,01 <br />
|0,067 <br />
|0,061 <br />
|1 <br />
|0,064 <br />
|6,4<br />
|-<br />
|09:01 <br />
|0,01 <br />
|0,056 <br />
|0,05 <br />
|1 <br />
|0,053 <br />
|5,3<br />
|-<br />
|01:09 <br />
|0,01 <br />
|0,074 <br />
|0,073 <br />
|1 <br />
|0,0735 <br />
|7,35<br />
|-<br />
|} <br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strain-Grafale2.png|800px]]<br />
|}<br />
<br />
<br />
{|<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Grafale1.png|400px]]<br />
|<!--column1-->[[File:Bsas2012-strains-grafico5.png|400px]]<br />
|}<br />
<br />
<br />
<br />
<br />
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.<br />
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.<br />
<br />
==== At the same initial OD: 0.2, followed over time ====<br />
<br />
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. <br />
<br />
{| align="center" <br />
|- valign="top"<br />
|<br />
{| class="wikitable"<br />
|+ Cultures set at initial OD: 0.2 and measured over time (Days 0 and 2)<br />
! scope="row" style="background: #7ac5e8"|Strain<br />
! scope="row" style="background: #7ac5e8"|Day 0 <br />
! scope="row" style="background: #7ac5e8"|Day 2<br />
|-<br />
|TCY 3190 (-H+T) <br />
|0,2<br />
|2,92<br />
|-<br />
|TCY 3265 (+H-T) <br />
|0,2<br />
|0,19<br />
|-<br />
|Coculture of strains (TCY 3190- TCY 3265) <br />
|0,2<br />
|2,76<br />
|-<br />
|Negative control (TCY 3043 / -H-T) <br />
|0,2<br />
|0,6<br />
|-<br />
|Positive Control (TCY 3154/ +H+T) <br />
|0,2<br />
|2,54<br />
|}<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-wednesday.png|500px]]<br />
|-<br />
|Picture: Day 1 after starting cultures, shows different OD reached by strains. <br />
|}<br />
<br />
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.<br />
<br />
== Coculture in Agar and Revertant mutation control ==<br />
<br />
<br />
Through this experiment we aim to quantify the rate of revertants of each strain. <br />
<br />
We used Petri dishes with agar medium with (+) and without (-) Trp and His as shown in the following table.<br />
<br />
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). <br />
At the same time, 3 controls (one for each strain) were carried in YPD complete medium to check the viability of each strain separately. <br />
<br />
{| class="wikitable"<br />
! scope="row" style="background: #7ac5e8"|Medium H<br />
! scope="row" style="background: #7ac5e8"|Medium T<br />
! scope="row" style="background: #7ac5e8"|Grass (10 6 cel) <br />
! scope="row" style="background: #7ac5e8"|Seed (102 cel) <br />
! scope="row" style="background: #7ac5e8"|Description of experiment <br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 1<br />
! scope="row" style="background: #7ac5e8"|Results after 3 days - Replica 2<br />
|-<br />
|(-) <br />
|(+) <br />
|(-) <br />
|Strain –H+T <br />
|Control of His revertants <br />
|7 <br />
|7<br />
|-<br />
|-<br />
|(+)<br />
|(-)<br />
|(-)<br />
|Strain +H-T <br />
|Control of Trp revertants <br />
|2 <br />
|7<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain +H-T<br />
|Strain –H+T<br />
|Coculture; we expect to see natural cooperation<br />
|960<br />
|800<br />
|-<br />
|(-)<br />
|(-)<br />
|Strain –H+T<br />
|Strain +H-T<br />
|Coculture; we expect to see natural cooperation<br />
|500<br />
|712<br />
|-<br />
|(-)<br />
|(-)<br />
|(-)<br />
|Strain +H+T<br />
|Viability of yeasts in medium<br />
|171<br />
|(-)<br />
|}<br />
<br />
'''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'''. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-placas2.jpg|300px]]<br />
|<!--column1-->[[File:Bsas2012-strains-placas1.jpg|300px]]<br />
|-<br />
|Petri Dishes<br />
| With marks of the counting of colonies<br />
|}<br />
<br />
<br />
==== Results ====<br />
The viability of the strains was high as expected as well as the viability of a control positive strain in the –H-T medium. <br />
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. <br />
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.<br />
<br />
== Measurement of Trp in medium and Basal Production ==<br />
<br />
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.<br />
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.<br />
<br />
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.<br />
<br />
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.<br />
<br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-Trpmario.png]]<br />
|-<br />
|Graph:Tryptophan calibration curve<br />
|}<br />
<br />
<br />
<br />
==== Results ====<br />
<br />
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.<br />
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.<br />
<br />
Because of time constraints, we haven't been able to check the method with either our designed strains nor the non-exporting ones.<br />
<br />
== Growth dependence on the Trp and His concentrations ==<br />
<br />
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. <br />
<br />
We used strain ACL-379, that is auxotroph for both Trp and His. <br />
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. <br />
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.<br />
<br />
{| class="wikitable"<br />
|+Growth of ACL-379 as a function of Trp and His concentration<br />
! scope="row" style="background: #7ac5e8"|Medium<br />
! scope="row" style="background: #7ac5e8"|OD Replica 1<br />
! scope="row" style="background: #7ac5e8"|OD Replica 2<br />
|-<br />
|SC<br />
|0,001<br />
|(-0,0036)<br />
|-<br />
|HLC <br />
|(-0,003)<br />
|(-0,019)<br />
|-<br />
|S1 HLC (1:10) <br />
|0,256 <br />
|0,217<br />
|-<br />
|S2 HLC (1:10) <br />
|0,301 <br />
|0,311<br />
|-<br />
|S3 HLC (1:10) <br />
|0,154 <br />
|0,155<br />
|-<br />
|S4 HLC <br />
|0,393 <br />
|0,409<br />
|-<br />
|S5 HLC <br />
|0,013 <br />
|0,003<br />
|-<br />
|TLC <br />
|(-0,008) <br />
|(-0,012)<br />
|-<br />
|S1 TLC (1:10) <br />
|0,368 <br />
|0,384<br />
|-<br />
|S2 TLC (1:10) <br />
|0,207 <br />
|0,2<br />
|-<br />
|S3 TLC (1:10) <br />
|0,117 <br />
|0,097<br />
|-<br />
|S4 TLC <br />
|0,47 <br />
|0,432<br />
|-<br />
|S5 TLC <br />
|0,238 <br />
|0,257<br />
|-<br />
|HTLC (1:10) <br />
|0,488 <br />
|0,491<br />
|} <br />
<br />
==== Results ====<br />
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. <br />
<br />
These results can also be observed by comparison of images that show the tubes at different OD. <br />
<br />
{| class="wikitable" border="1"<br />
|-<br />
|<!--column1-->[[File:Bsas2012-strains-alan1.png|500px]]<br />
|<!--column2-->[[File:Bsas2012-strains-ultima.jpg|500px]] <br />
|-<br />
|Images from HLC series<br />
|Images from TLC series<br />
|}<br />
<br />
Notes: <br />
SC: Synthetic complete medium with all the aminoacids. It was used as a blank for the spectrofluorometer.<br />
<br />
HTLC is the culture in the medium with all the required aminoacids.<br />
<br />
S(Number) are the serial dilutions of HTLC with medium that lacks Histidine (HLC) and Tryptophane (TLC.<br />
<br />
HLC and TLC are mediums without cells.</div>Abush84