Team:Buenos Aires/Results/BBsTesting

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= After the Jamboree! =
= After the Jamboree! =
-
When we returned from the LatinAmerican Jamboree we focused all our effords on completing the neccesary transformations to test our devices and our projet as a whole.  
+
 
 +
DEVICE TESTING
 +
 
 +
https://2012.igem.org/Team:Buenos_Aires/Results/DevicesTesting
 +
 
 +
 
 +
SYNECO TESTING
 +
 
 +
https://2012.igem.org/Team:Buenos_Aires/Results/SynEcoTesting
 +
 
 +
 
 +
 
 +
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.  
We devided this task in three sections
We devided this task in three sections
 +
== Week 1&2 : Yeast expression vectors & Transformations ==
== Week 1&2 : Yeast expression vectors & Transformations ==
-
xxoo
+
In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:
 +
# '''pCM182/5''', which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996]. 
 +
# '''pEG202''', with a 2 ori, HIS3 marker and a constitutive promoter (PADH1).
 +
 
 +
 
 +
{| style="width:100%"
 +
|[[File:BsAs2012-plasmid-PEG202.jpg|400px]]
 +
|[[File:BsAs2012-plasmid-BPCM185.gif|340px]]
 +
|}
 +
 
 +
The cloning we did was:
 +
 
 +
{| class="wikitable"
 +
|
 +
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792009</partinfo> (PoliHa)
 +
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792010</partinfo> (TRPZipper2)
 +
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792011</partinfo> (PoliHb)
 +
! scope="row" style="background: #7ac5e8"|<partinfo>BBa_K792012</partinfo> (PoliWb)
 +
|-
 +
! scope="row" style="background: #81BEF7"|pCM182 (TRPa)
 +
|
 +
! scope="row" style="background: #01DF01"|X
 +
|
 +
! scope="row" style="background: #01DF01"|X
 +
|-
 +
! scope="row" style="background: #81BEF7"|pCM185 (TRPb)
 +
|
 +
! scope="row" style="background: #01DF01"|X
 +
|
 +
! scope="row" style="background: #01DF01"|X
 +
|-
 +
! scope="row" style="background: #81BEF7"|pEG202 (HIS)
 +
! scope="row" style="background: #01DF01"|X
 +
|
 +
! scope="row" style="background: #01DF01"|X
 +
|
 +
|}
 +
 
 +
 
 +
==== Protocol ====
 +
 
 +
# Digestion of plasmids and TRP/HIS export device:
 +
##pCM182; pCM 185;  BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.
 +
##pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.
 +
# Purification of digested vectors (pCM185; pCM182; pEG202)
 +
# Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)
 +
# Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.
 +
# Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.
 +
# Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).
 +
 
 +
 
 +
Once obtained the desired constructions, we transformed yeast strains:
 +
# TCY3081: W303, bar1-, ura3::PAct1-YFP
 +
# TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405
 +
 
 +
 
 +
 
 +
We got the following '''transformed strains''':
{|
{|
-
|YFP_TRP+++_TRPZipper
 
-
| details
 
|
|
-
[[File:Bsas2012-Gl.png | 25px]]
+
{| style="width:50%"
 +
| rowspan="3" | [[File:BsAs2012-icono-Cepa1.jpg | 200px]]
 +
! scope="row" style="background: #7ac5e8"| Name
 +
| '''YFP_TRPa_TRPZipper2'''
|-
|-
-
|YFP_TRP+_TRPZipper
+
! scope="row" style="background: #CEE3F6"|Strain
-
| details
+
|TCY3081 (YFP)
 +
|-
 +
! scope="row" style="background: #CEE3F6"|Plasmid
 +
|pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)
 +
|}
|
|
-
[[File:Bsas2012-Gl.png | 25px]]
+
{| style="width:50%"
 +
| rowspan="3" | [[File:BsAs2012-icono-Cepa2.jpg | 200px]]
 +
! scope="row" style="background: #7ac5e8"| Name
 +
| '''YFP_TRPa_PoliWb'''
|-
|-
-
|YFP_TRP+++_PoliWb
+
! scope="row" style="background: #CEE3F6"|Strain
-
| details
+
|TCY3081 (YFP)
 +
|-
 +
! scope="row" style="background: #CEE3F6"|Plasmid
 +
|pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)
 +
|}
 +
|}
 +
 
 +
{|
|
|
-
[[File:Bsas2012-Gl.png | 25px]]
+
{| style="width:50%"
 +
| rowspan="3" | [[File:BsAs2012-icono-Cepa3.jpg | 200px]]
 +
! scope="row" style="background: #7ac5e8"| Name
 +
| '''YFP_TRPb_TRPZipper2'''
|-
|-
 +
! scope="row" style="background: #CEE3F6"|Strain
 +
|TCY3081 (YFP)
|-
|-
-
|YFP_TRP+_PoliWb
+
! scope="row" style="background: #CEE3F6"|Plasmid
-
| details
+
|pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)
 +
|}
|
|
-
[[File:Bsas2012-Gl.png | 25px]]
+
{| style="width:50%"
 +
| rowspan="3" | [[File:BsAs2012-icono-Cepa4.jpg | 200px]]
 +
! scope="row" style="background: #7ac5e8"| Name
 +
| '''YFP_TRPb_PoliWb'''
|-
|-
 +
! scope="row" style="background: #CEE3F6"|Strain
 +
|TCY3081 (YFP)
 +
|-
 +
! scope="row" style="background: #CEE3F6"|Plasmid
 +
|pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)
|}
|}
 +
|}
 +
 +
{|
 +
|
 +
{| style="width:50%"
 +
| rowspan="3" | [[File:BsAs2012-icono-Cepa6.jpg | 200px]]
 +
! scope="row" style="background: #7ac5e8"| Name
 +
| '''CFP_HIS_PoliHa'''
 +
|-
 +
! scope="row" style="background: #CEE3F6"|Strain
 +
|TCY3128 (CFP)
 +
|-
 +
! scope="row" style="background: #CEE3F6"|Plasmid
 +
|pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)
 +
|}
 +
|
 +
{| style="width:50%"
 +
| rowspan="3" | [[File:BsAs2012-icono-Cepa5.jpg | 200px]]
 +
! scope="row" style="background: #7ac5e8"| Name
 +
| '''CFP_HIS_PoliHb'''
 +
|-
 +
! scope="row" style="background: #CEE3F6"|Strain
 +
|TCY3128 (CFP)
 +
|-
 +
! scope="row" style="background: #CEE3F6"|Plasmid
 +
|pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)
 +
|}
 +
|}
 +
 +
== Week 3: Synthetic Ecology Characterization ==
 +
 +
Our task is to characterize our system including the devices functioning.
 +
We want specifically to: 
 +
 +
#Quantify the export of Trp as proof that the devices work
 +
#Determine experimentally some of the parameters that we used in the model
 +
#Characterize the growth of the transformed strains in coculture
 +
 +
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.
 +
 +
-
== Week 3: synthetic Ecology Characterization ==
+
=== Devices characterization ===
-
xxoo
+
-
=== BB characterization ===
 
-
xxoo
 
==== Secretion Rate of Trp as a function of culture growth  ====
==== Secretion Rate of Trp as a function of culture growth  ====
-
The aim of this experiment is to characterize Trp secretion on devices 2 and 4. We cultured the transformed strains overnight in 5 ml of medium +H-T until they reached an OD: 0.1 (exponential phase).
+
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?
-
In order to have a clear signal we used:
+
To test this we used the following strains:
-
TCY3081 <- pCM185 <- Device 2
+
{|
 +
|
 +
{|
 +
|-
 +
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]
 +
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]
 +
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]
 +
|- align="center"
 +
|YFP_TRPb_TRPZipper2
 +
|YFP_TRPb_PolyWb
 +
|YFP_TRPb
 +
|}
 +
| rowspan="2" |
 +
{|
 +
| [[File:BsAs2012-icono-YFP.jpg | 200px]]
 +
|- align="center"
 +
|YFP
 +
|}
-
TCY3081 <- pCM185 <- Device 4
+
|-
 +
|
 +
{|
 +
|-
 +
|[[File:BsAs2012-icono-Cepa1.jpg|200px]]
 +
|[[File:BsAs2012-icono-Cepa2.jpg|200px]]
 +
|[[File:BsAs2012-icono-YFP-182.jpg | 200px]]
 +
|- align="center"
 +
|YFP_TRPa_TRPZipper2
 +
|YFP_TRPa_PolyWb
 +
|YFP_TRPa
 +
|}
-
TCY3081 <- pCM185
+
|}
-
TCY3081 <- pCM182 <- Device 2
 
-
TCY3081 <- pCM182 <- Device 4
+
===== Protocol =====
-
TCY3081 <- pCM182
+
*We started  5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.
 +
*Starting OD for the assay 0.1 (exponential phase).
 +
*We measured OD every hour until they reached an OD: 0.8 (5 hs approximately).
 +
*We measured the Trp signal for each culture medium using the spectrofluorometer.
-
TCY3081
+
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.
 +
Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.
-
We did 3 replica of each culture. As from this point we measured OD every hour until they reached an OD: 0.8 (approximately). We then measured the Trp signal for each culture medium using the spectrofluorometer.
+
We used a simple model to measure the secretion rate for all the strains. Since the cultures are in exponential phase, we take
 +
[[File:BsAs2012-eqTrp1.jpg | 225px]]
-
==== Trp Secretion at different His concentrations in medium and His secretion at different Trp concentrations in medium  ====
+
After a few calculations, we find that
-
We started cultures in -T of the following strains:  
+
[[File:BsAs2012-eqTrp2.jpg | 225px]]
-
i. strain TCY3081+pCM185 containing device2
+
===== Results =====
-
ii. strain TCY3081+pCM185 containing device4
+
-
iii. strain TCY3081+pCM185 (empty plasmid)
+
-
We measured the OD of the cultures after 12 hs. We prepared cultures with increasing concentrations of His in medium (1X, 1/4; 1/8; 0X)and set cultures at initial OD: 0.1. After 5 hours we measured the final OD and Trp in medium.
+
Next we show the average OD for each strains used, needed to calculate the secretation rates of Trytophan.
 +
{|
 +
[[File:BsAs2012Odvstimea4.jpg | 350px]]
 +
|
 +
[[File:BsAs2012Odvstimeab.jpg | 350px]]
 +
|}
 +
Figure X. check
 +
 +
[[File:BsAs2012rate2.jpg| 350px]]
 +
 +
Figure X. check
 +
 +
==== Tryptophan secretion at increasing histidine concentrations ====
 +
 +
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.
 +
 +
In this experiment we used strains:
 +
 +
 +
{|
 +
|-
 +
|[[File:BsAs2012-icono-Cepa3.jpg|200px]]
 +
|[[File:BsAs2012-icono-Cepa4.jpg|200px]]
 +
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]
 +
|- align="center"
 +
|YFP_TRPb_TRPZipper2
 +
|YFP_TRPb_PolyWb
 +
|YFP_TRPb (control)
 +
|}
 +
 +
 +
 +
 +
 +
 +
 +
===== Protocol =====
 +
 +
# Starters of each strain used were grown over night in -T media, at 30 °C in shaker.
 +
# After 12 hs, cells were pelleted and washed with -HT media.
 +
# 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.
 +
# 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.
 +
 +
===== Results =====
 +
 +
[[File: BsAs2012TrpvHisvv.jpg|350px]]
=== Strain characterization ===
=== Strain characterization ===
-
==== Experimental determination of K death ====
+
==== Experimental determination of strains death rate====
-
In order to determinate the K death used in the modeling section of our wiki, we set cultures of the two auxotrophic strains without being transformed (TCY3081 and TCY3128) in medium –HT at an initial OD of 0.01.  
+
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.
-
Taking into account that an OD:1 is approximately 3.10 7 cells/ml, we plated 133 µl in order to have around 200 colonies in the first day. Each following 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.
+
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''.
 +
===== Protocol =====
 +
For this experiment we used
 +
{|
 +
|-
 +
|[[File:BsAs2012-icono-YFP.jpg|200px]]
 +
|[[File:BsAs2012-icono-CFP.jpg|200px]]
 +
|- align="center"
 +
|YFP Strain
 +
|CFP Strain
 +
|}
 +
*We set cultures of the two auxotroph strains without being transformed (YFP and CFP) in medium –HT at an initial OD of 0.01.
 +
*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.
 +
===== Result =====
-
TABLA CON EL NUMERO DE CELULAS POR DIA
+
{| class="wikitable"
 +
! scope="row" style="background: #7ac5e8" |Strain
 +
! scope="row" style="background: #7ac5e8" |Replica
 +
! scope="row" style="background: #7ac5e8" |Monday
 +
! scope="row" style="background: #7ac5e8" |Tuesday
 +
! scope="row" style="background: #7ac5e8" |Wednesday
 +
|-
 +
|CFP
 +
|1
 +
|260
 +
|320
 +
|285
 +
|-
 +
|CFP
 +
|2
 +
|267
 +
|314
 +
|76
 +
|-
 +
|CFP
 +
|3
 +
|413
 +
|362
 +
|278
 +
|-
 +
|YFP
 +
|1
 +
|230
 +
|316
 +
|688
 +
|-
 +
|YFP
 +
|2
 +
|291
 +
|194
 +
|524
 +
|-
 +
|YFP
 +
|3
 +
|449
 +
|344
 +
|725
 +
|}
 +
'''Table:''' Number of colonies counted per plate.
-
==== Growth rate vs [TRP] and [HIS] ====
+
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.
-
We made dilutions in series for increasing concentrations of Trp or His(1/32; 1/16; 1/8 ; 1/4; 1/2; 1X) in a -T or -H medium respectively. We made 2 replica of each concentration.  
+
[[File:Bsas2012kdeathcells.png| 500px]]
-
We set the cultures of the different strains at a low initial OD:0.001 approximately.
 
-
i. strain TCY3081 without device in each Trp concentration
+
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.
-
ii. strain TCY3128 without device in each His concentration
 
-
iii. strain TCY3081 without device in Synthetic Complete medium
+
{|
 +
|[[File:BsAs2012_celldeath.png | 100px]]
 +
|[[File:BsAs2012_celldeath2.png| 100px]]
 +
|
 +
|}
 +
FigureX.
-
iv. strain TCY3128 without device in Synthetic Complete medium
+
==== Growth dependence on the Trp and His concentration====
-
v. Control (Synthetic Complete medium)
+
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).
-
We measured OD reached by cultures 12 hs after started.  
+
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).
-
CONVERTIR ESTO EN UNA TABLA CON LOS RESULTADOS
+
===== Protocol=====
-
=== Co-Culture of strains ===
+
1. For this experiment we would use strains YFP and CFP (non-transformed, without devices).
 +
We started cultures of 5 ml of initial OD: 0.025 for:
-
We proceeded then to test the coculture growth of transformed strains with devices vs transformed strains with empty devices and non transformed strains.  
+
{| class="wikitable"
 +
| Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x
 +
|-
 +
| Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x
 +
|-
 +
| Strain YFP at Synthetic Complete media
 +
|-
 +
| Strain CFP at Synthetic Complete media
 +
|}
 +
 +
2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.
-
{| style="width: 100%"
+
===== Results =====
-
| align="center" | [[File:IGEM-Buenos Aires-Coculture.png]]
+
To be done with our strains soon.
 +
 
 +
=== Coculture of  transformed strains ===
 +
 
 +
We did the first experiment to test whether our system works and how two transformed strains grow together.
 +
 
 +
We designed an assay to do so; following the growth of these strains:
 +
 
 +
{|
 +
|-
 +
|[[File:BsAs2012-icono-CFP-202.jpg|200px]]
 +
|[[File:BsAs2012-icono-YFP-185.jpg|200px]]
 +
|- align="center"
 +
|CFP_His
 +
|YFP_TRPb
|}
|}
-
'''Table:''' Coculture planification. Number indicates level of priority of the experiment for characterization of devices.
 
-
Due to a time constrain, we ordered the experiments by level of priority and proceeded to test our devices by cocultures 1 and 2.
 
-
Starters of each strain were done in medium complementary to the auxotrophy of the strain so that they would maintain their plamids (medium –H for pEG202; medium –T for pCM182/5 and medium SC for cells without plasmid), then sonicated briefly in low power and then washed with medium –H-T . When they are in exponential phase, we set the coculture of strains was at OD 600: 0.02 (Total, 1:1), in 5 ml of medium –H-T.
+
{|
 +
|
 +
{| class="wikitable"
 +
|+ Transformed cells coculture and controls.
 +
|! scope="row" align="center" style="background: #7ac5e8"| '''Treatment'''
 +
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain A'''
 +
|! scope="row" align="center" style="background: #7ac5e8"| '''Strain B'''
 +
|-
 +
|1
 +
|YFP_TRPb_TRPZipper2
 +
|CFP_HIS_PolyHa
 +
|-
 +
|2
 +
|YFP_TRPb_TRPZipper2
 +
|CFP_HIS
 +
|-
 +
|3
 +
|YFP_TRPb_TRPZipper2
 +
|! scope="row" style="background: #CCCCCC"|
 +
|-
 +
|4
 +
|YFP_TRPb
 +
|CFP_HIS_PolyHa
 +
|-
 +
|5
 +
|! scope="row" style="background: #CCCCCC"|
 +
|CFP_HIS_PolyHa
 +
|-
 +
|6
 +
|YFP_TRPb
 +
|CFP_HIS
 +
|-
 +
|7
 +
|! scope="row" style="background: #CCCCCC"|
 +
|CFP_HIS
 +
|-
 +
|8
 +
|YFP_TRPb
 +
|! scope="row" style="background: #CCCCCC"|
 +
|}
 +
|}
-
We used epifluorescence microscope in order to determinate the strain proportion of each coculture. We used a 384 wells plate , with 20 µl of cyclohexamide 2x (CHX 2x) in each of the wells where we placed a sample. The density of the culture was calculated based on the cell density at in each wells.  
+
==== Protocol ====
 +
{|
 +
|
 +
# Starters of strains were grown over night at 30°C, according the scheme showed in the table
 +
# The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.
 +
# Cells were centrifugated and then washed with medium –HT.
 +
# We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1.
 +
# At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.
 +
# Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.
 +
# 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.
 +
|
 +
{|class="wikitable"
 +
|! scope="row" align="center" style="background: #7ac5e8"|'''Strain'''
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|! scope="row" align="center" style="background: #7ac5e8"|'''Media'''
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|-
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|YFP_TRPb_TRPZipper2
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|'''-T'''
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|-
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|YFP_TRPb
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|'''-T'''
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|-
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|CFP_HIS_PolyHa
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|'''-H'''
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|-
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|CFP_HIS
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|'''-H'''
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|}
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|}
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{| style="width: 100%"
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{| class="wikitable" style="width:50%"
| align="center" | [[File:Bsas2012-Wells.png‎|500px]]
| align="center" | [[File:Bsas2012-Wells.png‎|500px]]
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|-
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| 384 wells plate to be used for epifluorescence microscope.
|}
|}
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'''Graph:''' 384 wells plate to be used for epifluorescence microscope.
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==== Results ====
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[[File:BsAs2012coculture1.jpg | 600px]]
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{| style="width: 100%"
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[[File:BsAs2012coculture2.jpg| 600px]]
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| align="center" | [[File:Basas2012-expMinimo.png‎|500px]]
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|}
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'''Table:''' Coculture planification for number 1.
+

Latest revision as of 03:55, 27 October 2012

Contents

After the Jamboree!

DEVICE TESTING

https://2012.igem.org/Team:Buenos_Aires/Results/DevicesTesting


SYNECO TESTING

https://2012.igem.org/Team:Buenos_Aires/Results/SynEcoTesting


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.

We devided this task in three sections


Week 1&2 : Yeast expression vectors & Transformations

In order to construct the yeast expression plasmids we choosed 3 vectors, 2 with a tryptophan marker and 1 with an histidine marker:

  1. pCM182/5, which are centromeric plasmids with TRP1 marker, and with a doxycycline repressible promoter [Gari et al 1996].
  2. pEG202, with a 2 ori, HIS3 marker and a constitutive promoter (PADH1).


BsAs2012-plasmid-PEG202.jpg 340px

The cloning we did was:

<partinfo>BBa_K792009</partinfo> (PoliHa) <partinfo>BBa_K792010</partinfo> (TRPZipper2) <partinfo>BBa_K792011</partinfo> (PoliHb) <partinfo>BBa_K792012</partinfo> (PoliWb)
pCM182 (TRPa) X X
pCM185 (TRPb) X X
pEG202 (HIS) X X


Protocol

  1. Digestion of plasmids and TRP/HIS export device:
    1. pCM182; pCM 185; BBa_K792010 y BBa_K792012 were digested with BamHI and Pst1 restriction enzymes.
    2. pEG202; BBa_K792009 y BBa_K792011 were digested with BamHI and Not1.
  2. Purification of digested vectors (pCM185; pCM182; pEG202)
  3. Ligation of vectors and devices according the anterior table (T4 ligase protocol, overnight)
  4. Transformation of E. Coli DH5a with the ligation products. Bacterias were plated on LB-agar with Ampicillin, and incubated over night at 37 °C.
  5. Colonies were used for liquid cultures (LB + Ampicillin) and minipreps were made.
  6. Constructions (vector + insert) were checked by digestion with restriction enzymes, and 1%-agarose gel (1kb and 100bp as markers).


Once obtained the desired constructions, we transformed yeast strains:

  1. TCY3081: W303, bar1-, ura3::PAct1-YFP
  2. TCY3128: W303, bar1-, leu2:: Pprm1-CFP 405


We got the following transformed strains:

BsAs2012-icono-Cepa1.jpg Name YFP_TRPa_TRPZipper2
Strain TCY3081 (YFP)
Plasmid pCM182 (TRPa) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)
BsAs2012-icono-Cepa2.jpg Name YFP_TRPa_PoliWb
Strain TCY3081 (YFP)
Plasmid pCM182 (TRPa) + <partinfo>BBa_K792012</partinfo> (PoliWb)
BsAs2012-icono-Cepa3.jpg Name YFP_TRPb_TRPZipper2
Strain TCY3081 (YFP)
Plasmid pCM185 (TRPb) + <partinfo>BBa_K792010</partinfo> (TRPZipper2)
BsAs2012-icono-Cepa4.jpg Name YFP_TRPb_PoliWb
Strain TCY3081 (YFP)
Plasmid pCM185 (TRPb) + <partinfo>BBa_K792012</partinfo> (PoliWb)
BsAs2012-icono-Cepa6.jpg Name CFP_HIS_PoliHa
Strain TCY3128 (CFP)
Plasmid pEG202 (HIS) + <partinfo>BBa_K792009</partinfo> (PoliHa)
BsAs2012-icono-Cepa5.jpg Name CFP_HIS_PoliHb
Strain TCY3128 (CFP)
Plasmid pEG202 (HIS) + <partinfo>BBa_K792011</partinfo> (PoliHb)

Week 3: Synthetic Ecology Characterization

Our task is to characterize our system including the devices functioning. We want specifically to:

  1. Quantify the export of Trp as proof that the devices work
  2. Determine experimentally some of the parameters that we used in the model
  3. Characterize the growth of the transformed strains in coculture

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.


Devices characterization

Secretion Rate of Trp as a function of culture growth

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?

To test this we used the following strains:

BsAs2012-icono-Cepa3.jpg BsAs2012-icono-Cepa4.jpg BsAs2012-icono-YFP-185.jpg
YFP_TRPb_TRPZipper2 YFP_TRPb_PolyWb YFP_TRPb
BsAs2012-icono-YFP.jpg
YFP
BsAs2012-icono-Cepa1.jpg BsAs2012-icono-Cepa2.jpg BsAs2012-icono-YFP-182.jpg
YFP_TRPa_TRPZipper2 YFP_TRPa_PolyWb YFP_TRPa


Protocol
  • We started 5ml cultures with 3 replica until they reached exponential phase, overnight, using a -T medium.
  • Starting OD for the assay 0.1 (exponential phase).
  • We measured OD every hour until they reached an OD: 0.8 (5 hs approximately).
  • We measured the Trp signal for each culture medium using the spectrofluorometer.

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.

Therefore the secretion rates calculated will be higher than the actual ones. We used an empty plasmid as control to study tryptophan diffusion.

We used a simple model to measure the secretion rate for all the strains. Since the cultures are in exponential phase, we take

BsAs2012-eqTrp1.jpg

After a few calculations, we find that

BsAs2012-eqTrp2.jpg

Results

Next we show the average OD for each strains used, needed to calculate the secretation rates of Trytophan.

BsAs2012Odvstimea4.jpg

BsAs2012Odvstimeab.jpg

Figure X. check

BsAs2012rate2.jpg 

Figure X. check

Tryptophan secretion at increasing histidine concentrations

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.

In this experiment we used strains:


BsAs2012-icono-Cepa3.jpg BsAs2012-icono-Cepa4.jpg BsAs2012-icono-YFP-185.jpg
YFP_TRPb_TRPZipper2 YFP_TRPb_PolyWb YFP_TRPb (control)




Protocol
  1. Starters of each strain used were grown over night in -T media, at 30 °C in shaker.
  2. After 12 hs, cells were pelleted and washed with -HT media.
  3. 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.
  4. 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.
Results

BsAs2012TrpvHisvv.jpg

Strain characterization

Experimental determination of strains death rate

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.

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.

Protocol

For this experiment we used

BsAs2012-icono-YFP.jpg BsAs2012-icono-CFP.jpg
YFP Strain CFP Strain
  • We set cultures of the two auxotroph strains without being transformed (YFP and CFP) in medium –HT at an initial OD of 0.01.
  • 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.
Result
Strain Replica Monday Tuesday Wednesday
CFP 1 260 320 285
CFP 2 267 314 76
CFP 3 413 362 278
YFP 1 230 316 688
YFP 2 291 194 524
YFP 3 449 344 725

Table: Number of colonies counted per plate.

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.

Bsas2012kdeathcells.png


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.


100px 100px 
FigureX.

Growth dependence on the Trp and His concentration

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).

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).


Protocol

1. For this experiment we would use strains YFP and CFP (non-transformed, without devices). We started cultures of 5 ml of initial OD: 0.025 for:

Strain YFP at the following media [Trp] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x
Strain CFP at the following media [His] = 1x; 0.5x; 0.25x; 0.125x; 0.0625x, 0.03125x
Strain YFP at Synthetic Complete media
Strain CFP at Synthetic Complete media

2. Cultures would be left overnight (12 hs) at 30°C with agitation and we measured OD reached with the use of spectrophotometer.

Results

To be done with our strains soon.

Coculture of transformed strains

We did the first experiment to test whether our system works and how two transformed strains grow together.

We designed an assay to do so; following the growth of these strains:

BsAs2012-icono-CFP-202.jpg BsAs2012-icono-YFP-185.jpg
CFP_His YFP_TRPb


Transformed cells coculture and controls.
Treatment Strain A Strain B
1 YFP_TRPb_TRPZipper2 CFP_HIS_PolyHa
2 YFP_TRPb_TRPZipper2 CFP_HIS
3 YFP_TRPb_TRPZipper2
4 YFP_TRPb CFP_HIS_PolyHa
5 CFP_HIS_PolyHa
6 YFP_TRPb CFP_HIS
7 CFP_HIS
8 YFP_TRPb

Protocol

  1. Starters of strains were grown over night at 30°C, according the scheme showed in the table
  2. The next day cultures were sonicated briefly in low power, and OD was measured in order to check they were in exponential phase.
  3. Cells were centrifugated and then washed with medium –HT.
  4. We set the culture of strains at OD: 0.02 in 5 ml of medium –HT with 3 replica, according to Table 1.
  5. At 0, 1, 2, 3, 4, 5, 6, 7, 8 and 22 hours we took samples of 20 µl.
  6. Samples were placed in a 384 wells plate, with 20 µl of cyclohexamide 2x (final concentration 1x) in each of the wells.
  7. 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.
Strain Media
YFP_TRPb_TRPZipper2 -T
YFP_TRPb -T
CFP_HIS_PolyHa -H
CFP_HIS -H
Bsas2012-Wells.png
384 wells plate to be used for epifluorescence microscope.

Results

BsAs2012coculture1.jpg

BsAs2012coculture2.jpg

Retrieved from "http://2012.igem.org/Team:Buenos_Aires/Results/BBsTesting"