http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=MAP2012.igem.org - User contributions [en]2024-03-29T05:53:03ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/File:BsAs2012coculture3.jpgFile:BsAs2012coculture3.jpg2012-10-27T03:53:52Z<p>MAP: </p>
<hr />
<div></div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/SynEcoTestingTeam:Buenos Aires/Results/SynEcoTesting2012-10-27T03:53:09Z<p>MAP: /* Results */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
=SynEco Testing =<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first assay to test whether our system works and how two transformed strains grow together. <br />
<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====<br />
<br />
[[File:BsAs2012coculture3.jpg | 800px]]<br />
<br />
<br />
In the time framed we worked with, we werent able to see significant differences in the cell density. We are aware, based on our model predictions that there could be a lag time of days (1-3). This is also consistent with the results obtained in other experiments. We will repeat this test in a longer time period.<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 />
<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.</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/SynEcoTestingTeam:Buenos Aires/Results/SynEcoTesting2012-10-27T03:52:47Z<p>MAP: /* Results */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
=SynEco Testing =<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first assay to test whether our system works and how two transformed strains grow together. <br />
<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====<br />
<br />
[[File:BsAs2012coculture1.jpg | 800px]]<br />
<br />
<br />
In the time framed we worked with, we werent able to see significant differences in the cell density. We are aware, based on our model predictions that there could be a lag time of days (1-3). This is also consistent with the results obtained in other experiments. We will repeat this test in a longer time period.<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 />
<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.</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/SynEcoTestingTeam:Buenos Aires/Results/SynEcoTesting2012-10-27T03:50:05Z<p>MAP: /* Results */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
=SynEco Testing =<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first assay to test whether our system works and how two transformed strains grow together. <br />
<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====<br />
<br />
[[File:BsAs2012coculture1.jpg | 700px]]<br />
<br />
<br />
In the time framed we worked with, we werent able to see significant differences in the cell density. We are aware, based on our model predictions that there could be a lag time of days (1-3). This is also consistent with the results obtained in other experiments. We will repeat this test in a longer time period.<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 />
<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.</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/SynEcoTestingTeam:Buenos Aires/Results/SynEcoTesting2012-10-27T03:38:53Z<p>MAP: /* Coculture of transformed strains */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
=SynEco Testing =<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first assay to test whether our system works and how two transformed strains grow together. <br />
<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====<br />
<br />
[[File:BsAs2012coculture1.jpg | 600px]]<br />
<br />
[[File:BsAs2012coculture2.jpg| 600px]]<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 />
<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.</div>MAPhttp://2012.igem.org/Team:Buenos_AiresTeam:Buenos Aires2012-10-27T03:34:38Z<p>MAP: /* Welcome to Buenos Aires 2012 iGEM Wiki! */</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 />
<br />
|<br />
{| style="width: 100%"<br />
| align="center" | [[File:Bsas2012-The Team.png|400px]]<br />
|}<br />
<br />
|}<br />
<br />
== Impact in the media ==<br />
<br />
* "[http://www.eldia.com.ar/edis/20121022/alumnos-argentinos-primera-vez-mundial-biologia-informaciongeneral1.htm Alumnos argentinos, por primera vez en un “Mundial” de biología]" Diario el Día, 10/22/12.<br />
<br />
* "[http://www.diariohoy.net/accion-verNota-id-213948-titulo-Argentinos_clasificaron_para_el_concurso_mundial_de_biolog%C3%ADa_sint%C3%A9tica_ Argentinos clasificaron para el concurso mundial de biología sintética]" Diario Hoy, 10/22/12.<br />
<br />
* "[http://www.aptus.com.ar/noticia/2932-competencia-mundial-de-biologia-sintetica Competencia mundial de Biología Sintética]" Aptus Noticias educativas, 10/21/12.<br />
<br />
* "[http://www.telam.com.ar/nota/41479/ Argentinos clasificaron para el concurso mundial de biología sintética]" Agencia de noticias TELAM, 10/20/12.<br />
<br />
* "[http://www.tvperu.gob.pe/noticias/tecnologia/otros/38160-argentinos-clasificaron-para-el-concurso-mundial-de-biologia-sintetica.html Argentinos clasificaron para el concurso mundial de biología sintética]" TV Perú Noticias , 10/20/12.<br />
<br />
* "[http://www.agenciacyta.org.ar/2012/10/debut-y-exito-argentino-en-olimpiadas-de-biologia-sintetica/ Debut y éxito argentino en olimpiadas de biología sintética]" Nota en el portal CyTA. 10/19/12.<br />
<br />
* "[http://www.tomamateyavivate.com.ar/formacion-y-universidades-argentinas/debut-y-exito-argentino-en-olimpiadas-de-biologia-sintetica/ Debut y éxito argentino en olimpiadas de biología sintética]" Tomamateyavivate, 10/19/12.<br />
<br />
* "[http://www.conicet.gov.ar/?p=2827 Estudiantes argentinos pasan a la etapa final de un certamen mundial]" Nota en la página de noticias del CONICET, 10/19/2012.<br />
<br />
* "[http://www.pagina12.com.ar/diario/sociedad/3-206038-2012-10-21.html Elegidos del Conicet]" Página 12, 20/10/12.<br />
<br />
* "[http://blogs.scientificamerican.com/lab-rat/2012/09/09/igem-buenos-aires-synthetic-bacterial-communities/ iGEM Buenos Aires: Synthetic bacterial communities]" Lab Rat blog at scientificamerican.com 9/9/12.</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/DevicesTestingTeam:Buenos Aires/Results/DevicesTesting2012-10-27T03:32:42Z<p>MAP: /* Results */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
= Devices testing =<br />
<br />
== Experimental Setup ==<br />
<br />
=== Plasmids and BBs ===<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Cloning protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
=== Yeast strains ===<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
=== Final transformed strains ===<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
<br />
<br />
== Secretion Rate of Trp as a function of culture growth ==<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 250px]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 250px]]<br />
|}<br />
Figure1. check<br />
<br />
[[File:BsAs2012rate2.jpg| 450px]]<br />
<br />
Figure2. check<br />
<br />
== Tryptophan secretion at increasing histidine concentrations ==<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis1.jpg|350px]]<br />
<br />
Figure 3. Final tryptophan concentration as a function of histidine concentration in the media.<br />
<br />
<br />
With this result we can infer that tryptophan export has no clear dependence on histidine concentration in the media. Therefore, supporting the assumption of the model.<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:Trp-bsas2012.png | 250px]]<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.</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/DevicesTestingTeam:Buenos Aires/Results/DevicesTesting2012-10-27T03:31:59Z<p>MAP: /* Results */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
= Devices testing =<br />
<br />
== Experimental Setup ==<br />
<br />
=== Plasmids and BBs ===<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Cloning protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
=== Yeast strains ===<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
=== Final transformed strains ===<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
<br />
<br />
== Secretion Rate of Trp as a function of culture growth ==<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 250px]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 250px]]<br />
|}<br />
Figure1. check<br />
<br />
[[File:BsAs2012rate2.jpg| 450px]]<br />
<br />
Figure2. check<br />
<br />
== Tryptophan secretion at increasing histidine concentrations ==<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis1.jpg|350px]]<br />
<br />
FigureX. Final tryptophan concentration as a function of histidine concentration in the media.<br />
<br />
<br />
With this result we can infer that tryptophan export has no clear dependence on histidine concentration in the media. Therefore, supporting the assumption of the model.<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:Trp-bsas2012.png | 250px]]<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.</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/DevicesTestingTeam:Buenos Aires/Results/DevicesTesting2012-10-27T03:30:44Z<p>MAP: /* Results */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
<br />
<br />
= Devices testing =<br />
<br />
== Experimental Setup ==<br />
<br />
=== Plasmids and BBs ===<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Cloning protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
=== Yeast strains ===<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
=== Final transformed strains ===<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
<br />
<br />
== Secretion Rate of Trp as a function of culture growth ==<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 250px]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 250px]]<br />
|}<br />
Figure1. check<br />
<br />
[[File:BsAs2012rate2.jpg| 450px]]<br />
<br />
Figure2. check<br />
<br />
== Tryptophan secretion at increasing histidine concentrations ==<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis1.jpg|350px]]<br />
<br />
FigureX. Final tryptophan secretion as a function of histiden concentration in the media.<br />
<br />
In Figure X we can see is the final tryptophan concentration in the culture as a function of histidine concentration in the media, in . <br />
<br />
With this result we can infer that tryptophan export has no clear dependence on histidine concentration in the media. Therefore, supporting the assumption of the model.<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:Trp-bsas2012.png | 250px]]<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.</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:43:53Z<p>MAP: /* Result */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our project as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|350px]]<br />
<br />
[[File: BsAs2012Trpratio.jpg|350px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|350px]]<br />
<br />
=== Strain characterization ===<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 expect 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 '''...Alan 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 />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-CFP-202.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|CFP_His<br />
|YFP_TRPb<br />
|}<br />
<br />
<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:43:30Z<p>MAP: /* Result */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our project as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|350px]]<br />
<br />
[[File: BsAs2012Trpratio.jpg|350px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|350px]]<br />
<br />
=== Strain characterization ===<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 expect 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| 100px]]<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-CFP-202.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|CFP_His<br />
|YFP_TRPb<br />
|}<br />
<br />
<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/File:Bsas2012kdeathcells.pngFile:Bsas2012kdeathcells.png2012-10-27T02:41:16Z<p>MAP: </p>
<hr />
<div></div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:32:02Z<p>MAP: /* Result */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our project as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|350px]]<br />
<br />
[[File: BsAs2012Trpratio.jpg|350px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|350px]]<br />
<br />
=== Strain characterization ===<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-CFP-202.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|CFP_His<br />
|YFP_TRPb<br />
|}<br />
<br />
<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:27:37Z<p>MAP: /* Coculture of transformed strains */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our project as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|350px]]<br />
<br />
[[File: BsAs2012Trpratio.jpg|350px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|350px]]<br />
<br />
=== Strain characterization ===<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-CFP-202.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|CFP_His<br />
|YFP_TRPb<br />
|}<br />
<br />
<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:26:21Z<p>MAP: /* Coculture of transformed strains */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our project as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|350px]]<br />
<br />
[[File: BsAs2012Trpratio.jpg|350px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|350px]]<br />
<br />
=== Strain characterization ===<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-CFP-202.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|<br />
|<br />
|}<br />
<br />
<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:20:41Z<p>MAP: /* Protocol */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our project as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|350px]]<br />
<br />
[[File: BsAs2012Trpratio.jpg|350px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|350px]]<br />
<br />
=== Strain characterization ===<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:20:16Z<p>MAP: /* Protocol */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our project as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|350px]]<br />
<br />
[[File: BsAs2012Trpratio.jpg|350px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|350px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<br />
<br />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:16:39Z<p>MAP: /* Protocol */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our project as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the aminoacid secreted by the device and the one diffused. <br />
<br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|450px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|450px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<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 />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:15:47Z<p>MAP: /* After the Jamboree! */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our project as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|450px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|450px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<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 />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:07:38Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|450px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|450px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<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 />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:07:05Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
===== Protocol =====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
===== Results =====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb (control)<br />
|}<br />
<br />
|}<br />
<br />
<br />
<br />
<br />
<br />
===== Protocol =====<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
===== Results =====<br />
<br />
[[File: BsAs2012TrpvHis.jpg|450px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|450px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<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 />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:04:19Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
==== Results ====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
{| class="wikitable" <br />
|YFP_TRPb_TRPZipper2<br />
|[[File:BsAs2012-icono-Cepa3.jpg| 200px]]<br />
|-<br />
|YFP_TRPb_PolyWb<br />
|[[File:BsAs2012-icono-Cepa4.jpg| 200px]]<br />
|-<br />
|YFP_TRPb (control)<br />
|[[File:BsAs2012-icono-YFP-185.jpg| 200px]]<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg|450px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|450px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<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 />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:03:54Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
==== Results ====<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
{| class="wikitable" <br />
|YFP_TRPb_TRPZipper2<br />
|[[File:BsAs2012-icono-Cepa3.jpg| 100px]]<br />
|-<br />
|YFP_TRPb_PolyWb<br />
|[[File:BsAs2012-icono-Cepa4.jpg| 100px]]<br />
|-<br />
|YFP_TRPb (control)<br />
|[[File:BsAs2012-icono-YFP-185.jpg| 100px]]<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg|450px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|450px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<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 />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:01:21Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa6.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa5.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
YFP_TRPb_TRPZipper2<br />
[[File:BsAs2012-icono-Cepa3.jpg| 100px]]<br />
<br />
YFP_TRPb_PolyWb<br />
[[File:BsAs2012-icono-Cepa4.jpg| 100px]]<br />
<br />
YFP_TRPb (control)<br />
[[File:BsAs2012-icono-YFP-185.jpg| 100px]]<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg|450px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|450px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<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 />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T02:00:30Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa6.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa5.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine whether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
[[File:BsAs2012-icono-Cepa3.jpg| 100px]]<br />
<br />
- YFP_TRPb_PolyWb<br />
[[File:BsAs2012-icono-Cepa4.jpg| 100px]]<br />
<br />
- YFP_TRPb (control)<br />
[[File:BsAs2012-icono-YFP-185.jpg| 100px]]<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg|450px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|450px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<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 />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:59:37Z<p>MAP: /* Results */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa6.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa5.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
[[File:BsAs2012-icono-Cepa3.jpg| 100px]]<br />
<br />
- YFP_TRPb_PolyWb<br />
[[File:BsAs2012-icono-Cepa4.jpg| 100px]]<br />
<br />
- YFP_TRPb (control)<br />
[[File:BsAs2012-icono-YFP-185.jpg| 100px]]<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg|450px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|450px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<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 />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done with our strains soon.<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:58:28Z<p>MAP: /* Experimental determination of strains death rate */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa6.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa5.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
[[File:BsAs2012-icono-Cepa3.jpg| 100px]]<br />
<br />
- YFP_TRPb_PolyWb<br />
[[File:BsAs2012-icono-Cepa4.jpg| 100px]]<br />
<br />
- YFP_TRPb (control)<br />
[[File:BsAs2012-icono-YFP-185.jpg| 100px]]<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg|450px]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg|450px]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<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 />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:57:03Z<p>MAP: /* Experimental determination of strains death rate */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa6.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa5.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
[[File:BsAs2012-icono-Cepa3.jpg| 100px]]<br />
<br />
- YFP_TRPb_PolyWb<br />
[[File:BsAs2012-icono-Cepa4.jpg| 100px]]<br />
<br />
- YFP_TRPb (control)<br />
[[File:BsAs2012-icono-YFP-185.jpg| 100px]]<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 200px]]<br />
<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 />
<br />
'''Result'''<br />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:56:33Z<p>MAP: /* Experimental determination of strains death rate */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
<br />
{|<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPb_TRPZipper2<br />
|YFP_TRPb_PolyWb<br />
|YFP_TRPb<br />
|}<br />
| rowspan="2" |<br />
{|<br />
| [[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
|- align="center"<br />
|YFP<br />
|}<br />
<br />
|-<br />
|<br />
{|<br />
|-<br />
|[[File:BsAs2012-icono-Cepa6.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa5.jpg|200px]]<br />
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]<br />
|- align="center"<br />
|YFP_TRPa_TRPZipper2<br />
|YFP_TRPa_PolyWb<br />
|YFP_TRPa<br />
|}<br />
<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
{|<br />
[[File:BsAs2012Odvstimea4.jpg | 150]]<br />
|<br />
[[File:BsAs2012Odvstimeab.jpg | 150]]<br />
|}<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
[[File:BsAs2012-icono-Cepa3.jpg| 100px]]<br />
<br />
- YFP_TRPb_PolyWb<br />
[[File:BsAs2012-icono-Cepa4.jpg| 100px]]<br />
<br />
- YFP_TRPb (control)<br />
[[File:BsAs2012-icono-YFP-185.jpg| 100px]]<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
[[File:BsAs2012-icono-YFP.jpg | 100px]]<br />
[[File:BsAs2012-icono-CFP.jpg | 100px]]<br />
<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 />
<br />
'''Result'''<br />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:53:55Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Strain<br />
|Name<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|300px]]<br />
|YFP_TRPb_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPb_PolyWb<br />
|-<br />
|[[ |300px]]<br />
|YFP_TRPb<br />
|-<br />
|<br />
|YFP_TRPa_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPa_PolyWb<br />
|-<br />
|<br />
|YFP_TRPa<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
[[File:BsAs2012Odvstime4.jpg | 150]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
[[File:BsAs2012-icono-Cepa3.jpg| 100px]]<br />
<br />
- YFP_TRPb_PolyWb<br />
[[File:BsAs2012-icono-Cepa4.jpg| 100px]]<br />
<br />
- YFP_TRPb (control)<br />
[[File:BsAs2012-icono-YFP-185.jpg| 100px]]<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
<br />
'''Result'''<br />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:53:00Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Strain<br />
|Name<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|300px]]<br />
|YFP_TRPb_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPb_PolyWb<br />
|-<br />
|[[ |300px]]<br />
|YFP_TRPb<br />
|-<br />
|<br />
|YFP_TRPa_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPa_PolyWb<br />
|-<br />
|<br />
|YFP_TRPa<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
[[File:BsAs2012Odvstime4.jpg | 150]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
[[File:BsAs2012-icono-Cepa3.jpg| 100px]]<br />
<br />
- YFP_TRPb_PolyWb<br />
[[File:BsAs2012-icono-Cepa2.jpg| 100px]]<br />
<br />
- YFP_TRPb (control)<br />
[[File:BsAs2012-icono-YFP-185.jpg | 100px]]<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
<br />
'''Result'''<br />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:50:04Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Strain<br />
|Name<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|300px]]<br />
|YFP_TRPb_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPb_PolyWb<br />
|-<br />
|[[ |300px]]<br />
|YFP_TRPb<br />
|-<br />
|<br />
|YFP_TRPa_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPa_PolyWb<br />
|-<br />
|<br />
|YFP_TRPa<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
[[File:BsAs2012Odvstime4.jpg | 150]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
[[File:BsAs2012-icono-Cepa1.jpg| 100px]]<br />
<br />
- YFP_TRPb_PolyWb<br />
[[File:BsAs2012-icono-Cepa2.jpg| 100px]]<br />
<br />
- YFP_TRPb (control)<br />
[[File:BsAs2012-icono-YFP-185.jpg | 100px]]<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
<br />
'''Result'''<br />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:49:21Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Strain<br />
|Name<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|300px]]<br />
|YFP_TRPb_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPb_PolyWb<br />
|-<br />
|[[ |300px]]<br />
|YFP_TRPb<br />
|-<br />
|<br />
|YFP_TRPa_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPa_PolyWb<br />
|-<br />
|<br />
|YFP_TRPa<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
[[File:BsAs2012Odvstime4.jpg | 150]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
[[File:BsAs2012-icono-Cepa1.jpg]]<br />
<br />
- YFP_TRPb_PolyWb<br />
[[File:BsAs2012-icono-Cepa2.jpg]]<br />
<br />
- YFP_TRPb (control)<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
<br />
'''Result'''<br />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:44:34Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Strain<br />
|Name<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|300px]]<br />
|YFP_TRPb_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPb_PolyWb<br />
|-<br />
|[[ |300px]]<br />
|YFP_TRPb<br />
|-<br />
|<br />
|YFP_TRPa_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPa_PolyWb<br />
|-<br />
|<br />
|YFP_TRPa<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
[[File:BsAs2012Odvstime4.jpg | 150]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
File:BsAs2012-icono-Cepa1.jpg<br />
<br />
- YFP_TRPb_PolyWb<br />
File:BsAs2012-icono-Cepa2.jpg<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
<br />
'''Result'''<br />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
[[File:BsAs2012-icono-YFP-185.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:42:13Z<p>MAP: /* Secretion Rate of Trp as a function of culture growth */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Strain<br />
|Name<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|300px]]<br />
|YFP_TRPb_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPb_PolyWb<br />
|-<br />
|[[ |300px]]<br />
|YFP_TRPb<br />
|-<br />
|<br />
|YFP_TRPa_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPa_PolyWb<br />
|-<br />
|<br />
|YFP_TRPa<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
[[File:BsAs2012Odvstime4.jpg | 150]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
<br />
'''Result'''<br />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T01:38:53Z<p>MAP: /* Secretion Rate of Trp as a function of culture growth */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
<br />
<br />
We got the following '''transformed strains''':<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPa_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_TRPZipper2'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''YFP_TRPb_PoliWb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3081 (YFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|}<br />
|}<br />
<br />
{|<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHa'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)<br />
|}<br />
|<br />
{| style="width:50%"<br />
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]<br />
! scope="row" style="background: #7ac5e8"| Name<br />
| '''CFP_HIS_PoliHb'''<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Strain<br />
|TCY3128 (CFP)<br />
|-<br />
! scope="row" style="background: #CEE3F6"|Plasmid<br />
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)<br />
|}<br />
|}<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the tryptophan devices- actually secrete tryptophan into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
[[File:BsAs2012-icono-YFP.jpg | 200px]]<br />
<br />
<br />
{| class="wikitable"<br />
|-<br />
|Strain<br />
|Name<br />
|-<br />
|[[File:BsAs2012-icono-Cepa1.jpg|300px]]<br />
|YFP_TRPb_TRPZipper2<br />
|-<br />
|[[File:BsAs2012-icono-Cepa2.jpg|300px]]<br />
|YFP_TRPb_PolyWb<br />
|-<br />
|<br />
|<br />
|}<br />
<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
NOTE: The fluorescence meausurements taken for the Tryptophan in the medium, take into account both the Amino Acid secreted by the device and the one diffused. <br />
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study Tryptophan diffusion.<br />
<br />
We used a simple model to measure the secretation rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
'''Results'''<br />
<br />
Next we show the average OD for each strains used, needed to calculate the export rates of Trytophan.<br />
<br />
[[File:BsAs2012Odvstime4.jpg | 150]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg| 150]]<br />
<br />
Figure X. check<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
<br />
'''Result'''<br />
<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 expect 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 />
FOTOS DE LAS PLACAS AQUIIII!<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan 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 it would requires more time than 3 days to observe significative cell dying.<br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
[[File:BsAs2012-icono-CFP-202.jpg | 200px]]<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T00:36:03Z<p>MAP: /* Protocol */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
{|<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 expect 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 />
<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''. <br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T00:29:53Z<p>MAP: /* Growth dependence on the Trp and His concentration */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
{|<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 expect 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 />
<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''. <br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing in order to characterize the system is the dependence of the growth rate on the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T00:10:03Z<p>MAP: /* Protocol */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
{|<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 expect 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 />
<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''. <br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing characterize the system is the dependence of the growth rate of the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
|-<br />
| Strain YFP at Synthetic Complete media<br />
|-<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T00:09:20Z<p>MAP: /* Protocol */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
{|<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 expect 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 />
<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''. <br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing characterize the system is the dependence of the growth rate of the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
<br />
<br />
{| class="wikitable"<br />
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
| Strain YFP at Synthetic Complete media<br />
| Strain CFP at Synthetic Complete media<br />
|}<br />
<br />
<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T00:06:26Z<p>MAP: /* Growth dependence on the Trp and His concentration */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate2.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
{|<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 expect 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 />
<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''. <br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An important thing characterize the system is the dependence of the growth rate of the culture with the concentration of the crossfeeding aminoacids, tryptophane (Trp) and histidine (His).<br />
<br />
This would allow us to estimate one of the parameters used in our model (the EC50, which is the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T00:03:11Z<p>MAP: /* Protocol */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
{|<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 expect 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 />
<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''. <br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An 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).<br />
<br />
This would allow us to estimate one of the parameters used in our model (EC50, the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices).<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T00:02:01Z<p>MAP: /* Experimental determination of strains death rate */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
{|<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 expect 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 />
<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''. <br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An 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).<br />
<br />
This would allow us to estimate one of the parameters used in our model (EC50, the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices) which are auxotroph for His and Trp.<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T00:01:34Z<p>MAP: /* Experimental determination of strains death rate */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
{|<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 expect 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 />
<br />
<br />
<br />
We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''. <br />
<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An 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).<br />
<br />
This would allow us to estimate one of the parameters used in our model (EC50, the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices) which are auxotroph for His and Trp.<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-27T00:00:36Z<p>MAP: /* Experimental determination of strains death rate */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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 />
*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 />
{|<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 expect 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. We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''.<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An 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).<br />
<br />
This would allow us to estimate one of the parameters used in our model (EC50, the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices) which are auxotroph for His and Trp.<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-26T23:58:50Z<p>MAP: /* Experimental determination of strains death rate */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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, we'll 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 />
*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 />
{|<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 expect 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. We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''.<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An 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).<br />
<br />
This would allow us to estimate one of the parameters used in our model (EC50, the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices) which are auxotroph for His and Trp.<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-26T23:58:04Z<p>MAP: /* Experimental determination of strains death rate */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<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, we'll 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 />
*We set cultures of the two auxotrophic 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 />
{|<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 expect 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. We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''.<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An 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).<br />
<br />
This would allow us to estimate one of the parameters used in our model (EC50, the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices) which are auxotroph for His and Trp.<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-26T23:56:10Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPb_TRPZipper2<br />
<br />
- YFP_TRPb_PolyWb<br />
<br />
- YFP_TRPb (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<br />
==== Experimental determination of strains death rate====<br />
<br />
We set out to determine how long can auxotrophic 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, we'll 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 />
*We set cultures of the two auxotrophic 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 />
{|<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 expect 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. We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''.<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An 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).<br />
<br />
This would allow us to estimate one of the parameters used in our model (EC50, the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices) which are auxotroph for His and Trp.<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-26T23:54:09Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
In this experiment we used strains: <br />
<br />
- YFP_TRPa_TRPZipper2<br />
- YFP_TRPb_TRPZipper2<br />
- YFP_TRPa (control)<br />
<br />
<br />
'''Protocol'''<br />
<br />
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<br />
==== Experimental determination of strains death rate====<br />
<br />
We set out to determine how long can auxotrophic 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, we'll 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 />
*We set cultures of the two auxotrophic 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 />
{|<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 expect 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. We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''.<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An 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).<br />
<br />
This would allow us to estimate one of the parameters used in our model (EC50, the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices) which are auxotroph for His and Trp.<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-26T23:29:11Z<p>MAP: /* Tryptophan secretion at increasing histidine concentrations */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
We got the following transformed strains:<br />
<br />
{| class="wikitable"<br />
| Name<br />
| Yeast used<br />
| Plasmid<br />
|}<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine wether the secretion of tryptophan depends on the concentration of another aminoacid in the media, such as histidine and what would be the necessary amount of histidine in medium for the start of our system. <br />
<br />
'''Protocol'''<br />
<br />
# Starters of YFP_TRPa_TRPZipper2, YFP_TRPb_TRPZipper2 and YFP_TRPa (control) strains were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHisbypromotor.jpg]]<br />
<br />
=== Strain characterization ===<br />
==== Experimental determination of strains death rate====<br />
<br />
We set out to determine how long can auxotrophic 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, we'll 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 />
*We set cultures of the two auxotrophic 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 />
{|<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 expect 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. We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''.<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An 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).<br />
<br />
This would allow us to estimate one of the parameters used in our model (EC50, the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices) which are auxotroph for His and Trp.<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAPhttp://2012.igem.org/Team:Buenos_Aires/Results/BBsTestingTeam:Buenos Aires/Results/BBsTesting2012-10-26T23:21:19Z<p>MAP: /* Week 3: Synthetic Ecology Characterization */</p>
<hr />
<div>{{:Team:Buenos_Aires/Templates/menu}}<br />
= After the Jamboree! =<br />
<br />
When we returned from the Latin America Jamboree we focused all our efforts on completing the neccesary transformations to test our devices and our projet as a whole. <br />
<br />
We devided this task in three sections<br />
<br />
== Week 1&2 : Yeast expression vectors & Transformations ==<br />
<br />
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:<br />
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. <br />
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1). <br />
<br />
<br />
{| style="width:100%"<br />
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]<br />
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]<br />
|}<br />
<br />
<br />
<br />
The cloning we did was:<br />
<br />
{| class="wikitable"<br />
|<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)<br />
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|-<br />
! scope="row" style="background: #81BEF7"|pEG202 (HIS)<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
! scope="row" style="background: #01DF01"|X<br />
|<br />
|}<br />
<br />
<br />
==== Protocol ====<br />
<br />
# Digestion of plasmids and TRP/HIS export device:<br />
##pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.<br />
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.<br />
# Purification of digested vectors (pCM185; pCM182; pEG202) <br />
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)<br />
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with ampicilin, and incubated over night at 37 °C.<br />
# Colonies were used for liquid cultures (LB + Ampicilin) and minipreps were made.<br />
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).<br />
<br />
<br />
Once obtained the desired constructions, we transformed yeast strains:<br />
# TCY3081: W303, bar1-, ura3::PAct1-YFP<br />
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405<br />
<br />
TCY3081 (YFP) TCY3128 (CFP) Strain Name<br />
pCM182 (TRPa) + BBa_K792010 (TRPZipper2) X YFP_TRPa_TRPZipper2<br />
pCM182 (TRPa) + BBa_K792012 (PoliWb) X YFP_TRPa_PoliWb<br />
pCM185 (TRPb) + BBa_K792010 (TRPZipper2) X YFP_TRPb_TRPZipper2<br />
pCM185 (TRPb) + BBa_K792012 (PoliWb) X YFP_TRPb_PoliWb<br />
pEG202 (HIS) + BBa_K792009 (PoliHa) X CFP_HIS_PoliHa<br />
pEG202 (HIS) + BBa_K792011 (PoliHb) X CFP_HIS_PoliHb<br />
<br />
== Week 3: Synthetic Ecology Characterization ==<br />
<br />
Our task is to characterize our system including the devices functioning.<br />
We want specifically to: <br />
<br />
#Quantify the export of Trp as proof that the devices work<br />
#Determine experimentally some of the parameters that we used in the model<br />
#Characterize the growth of the transformed strains in coculture<br />
<br />
In order to characterize the devices that we used to transform our strains we came up with the series of assays that we describe below.<br />
<br />
=== Devices characterization ===<br />
<br />
==== Secretion Rate of Trp as a function of culture growth ====<br />
<br />
The first step was to actually check if the construct works: do the transformed yeast strains - with the TRP BB - actually secrete TRP into the medium?<br />
<br />
To test this we used the following strains:<br />
<br />
*YFP_TRPb_TRPZipper2<br />
*YFP_TRPb_PolyWb<br />
*YFP_TRPb<br />
*YFP_TRPa_TRPZipper2<br />
*YFP_TRPa_PolyWb<br />
*YFP_TRPa<br />
*YFP<br />
<br />
'''Protocol'''<br />
<br />
*We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.<br />
*Starting OD for the assay 0.1 (exponential phase). <br />
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately). <br />
*We measured the Trp signal for each culture medium using the spectrofluorometer.<br />
<br />
<br />
We used a simple model to measure the export rate for all the strains. Since the cultures are in exponential phase, we take<br />
<br />
[[File:BsAs2012-eqTrp1.jpg | 225px]]<br />
<br />
After a few calculations, we find that<br />
<br />
[[File:BsAs2012-eqTrp2.jpg | 225px]]<br />
<br />
Next we show the average temporal evolution for each strains used, <br />
<br />
[[File:BsAs2012Odvstime4.jpg]]<br />
<br />
Figure X. check<br />
<br />
[[File:BsAs2012rate.jpg]]<br />
<br />
==== Tryptophan secretion at increasing histidine concentrations ====<br />
<br />
We asked ourselves which was the dependance of tryptophan secretion on the amount of histidine in medium. We carried on this test in order to determine if wether the secretion of tryptophan depends of the concentretation of another amino acid in media, such as histidine, or not. ¿the necessary amount of histidine in medium for the start of our system?<br />
<br />
'''Protocol'''<br />
<br />
# Starters of YFP_TRPa_TRPZipper2, YFP_TRPb_TRPZipper2 and YFP_TRPa (control) strains were grown over night in -T media, at 30 °C in shaker.<br />
# After 12 hs, cells were pelleted and washed with -HT media.<br />
# Cultures with increasing concentrations of histidine (0X, 1X, 1/4X and 1/16X) were set at an initial OD: 0.1, for each strain with 2 replica.<br />
# We left the cultures in shaker at 30ºC for 5 hours. After that time, we measured the final OD reached by cultures with the use of a spectrophotometer and the amount of tryptophan present in medium with a spectrofluorometer.<br />
<br />
'''Results'''<br />
<br />
[[File: BsAs2012TrpvHis.jpg]]<br />
<br />
[[File: BsAs2012ratioTrpvHis.jpg]]<br />
<br />
=== Strain characterization ===<br />
==== Experimental determination of strains death rate====<br />
<br />
We set out to determine how long can auxotrophic 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, we'll 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 />
*We set cultures of the two auxotrophic 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 />
{|<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 expect 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. We can infer from this data that though they have not died, they may have enter into a '''...Alan state'''.<br />
<br />
{|<br />
|[[File:BsAs2012_celldeath.png | 100px]]<br />
|[[File:BsAs2012_celldeath2.png| 100px]]<br />
|<br />
|}<br />
FigureX.<br />
<br />
==== Growth dependence on the Trp and His concentration====<br />
<br />
An 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).<br />
<br />
This would allow us to estimate one of the parameters used in our model (EC50, the amount of aminoacid at which the culture reaches half of the maximum growth).<br />
<br />
<br />
===== Protocol=====<br />
<br />
1. We would use strains YFP and CFP (non-transformed, without devices) which are auxotroph for His and Trp.<br />
We started cultures of 5 ml of initial OD: 0.025 for:<br />
<br />
- Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x<br />
<br />
- Strain YFP at Synthetic Complete media<br />
<br />
- Strain CFP at Synthetic Complete media<br />
<br />
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.<br />
<br />
===== Results ===== <br />
To be done!<br />
<br />
=== Coculture of transformed strains ===<br />
<br />
We did the first experiment to test whether our system works and how two transformed strains grow together.<br />
<br />
We designed an assay to do so; following the growth of these strains:<br />
<br />
{|<br />
|<br />
{| class="wikitable"<br />
|+ Transformed cells coculture and controls.<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''<br />
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''<br />
|-<br />
|1<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS_PolyHa<br />
|-<br />
|2<br />
|YFP_TRPb_TRPZipper2<br />
|CFP_HIS<br />
|-<br />
|3<br />
|YFP_TRPb_TRPZipper2<br />
|! scope="row" style="background: #CCCCCC"|<br />
|-<br />
|4<br />
|YFP_TRPb<br />
|CFP_HIS_PolyHa<br />
|-<br />
|5<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS_PolyHa<br />
|-<br />
|6<br />
|YFP_TRPb<br />
|CFP_HIS<br />
|-<br />
|7<br />
|! scope="row" style="background: #CCCCCC"|<br />
|CFP_HIS<br />
|-<br />
|8<br />
|YFP_TRPb<br />
|! scope="row" style="background: #CCCCCC"|<br />
|}<br />
|}<br />
<br />
==== Protocol ====<br />
{|<br />
|<br />
# Starters of strains were grown over night at 30°C, according the scheme showed in the table<br />
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.<br />
# Cells were centrifugated and then washed with medium –HT.<br />
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1. <br />
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.<br />
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.<br />
# We used epifluorescence microscope in order to determinate the strain proportion of each coculture. The density of the culture was calculated based on the cell density at in each wells.<br />
|<br />
{|class="wikitable"<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''<br />
|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''<br />
|-<br />
|YFP_TRPb_TRPZipper2<br />
|'''-T'''<br />
|-<br />
|YFP_TRPb<br />
|'''-T'''<br />
|-<br />
|CFP_HIS_PolyHa<br />
|'''-H'''<br />
|-<br />
|CFP_HIS<br />
|'''-H'''<br />
|}<br />
|}<br />
<br />
{| class="wikitable" style="width:50%"<br />
| align="center" | [[File:Bsas2012-Wells.png|500px]]<br />
|-<br />
| 384 wells plate to be used for epifluorescence microscope.<br />
|}<br />
<br />
<br />
==== Results ====</div>MAP